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Uptake of 5-hydroxytryptamine in rat isolated atria
Gen. Pharmac. Vol. 23, No. 4, pp. 613-617, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
UPTAKE OF 5 - H Y D R O X Y T R Y P T A M I N E IN RAT ISOLATED ATRIA CHARLES EL RAWADI,* MICHI~LE HEIMBURGER, MONIQUE DAVY, MICHI~LEMIDOL-MONNET, FRANqOlSE BESLOTand YvES COHEN Laboratoire de Pharmacologie, Facult~ de Pharmacie, F-92296 Ch~tenay-Malabry, France [Fax 4683-1303] (Received 14 January 1992) AI~--I. The mode of 5-hydroxytryptamine (5-HT) uptake by the rat isolated atria was studied and compared to noradrenaline (NA) uptake. 2. Rat isolated atria were incubated with ~'C-5-HT (46/aM) or 3H-NA (0.4/aM). After washing, the radioactivity fixed in atria was counted and the total NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents were measured by HPLC. 3. ~4C-5-HT uptake was reduced in atria from 6-hydroxydopamine (100mg/kg, i.p., 48hr before experiments) or reserpine (2.5 mg/kg, i.p., 24 and 48 hr before experiments) pretreated rats. 4. The incubation of atria with 5-HT (50/aM) at the same time as 3H-NA reduced the 3H-NA value fixed. 5. Addition of desipramine (I/aM) or hydrocortisone (150/aM) before the incubation of atria with ~4C-5-HT was without any effect on ~4C-5-HT uptake. In contrast, fluvoxamine (1/aM) or indalpine (5/aM) caused a slight inhibition. 6. These data indicate that 5-HT is taken into the NA storage vesicles within the atria sympathetic nerves. This uptake does not use the NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation was noticed and a part of it is intracellular.
INTRODUCTION In physiological conditions, there is no circulating 5-hydroxytryptamine (5-HT) because it is removed by platelets or vascular endothelium (Stahl and Meltzer, 1978; Shepro et al., 1975). In various pathological conditions, an excess of 5-HT is released from platelets and saturates the uptake by nonaggregating platelets and endothelium. These conditions are observed in the coronary vasospasm of Prinzmetars angina (Angus et al., 1982; Bove and Dewey, 1983), cerebral vasospasm after subarachnoid haemorrhage (Allen et al., 1974), digital vasospasm o f R a y n a u d ' s phenomenon (Halpern et al., 1960; Stranden et al., 1982) and migraine (Raskin,1981). The excess of 5-HT may then reach the heart, the vascular smooth muscle cells and/or sympathetic nerve terminals to elicit its different responses. The uptake of 5-HT out of serotonergic structures and the mechanism of this process were examined by different authors in various preparations. 5-HT is taken up and stored in noradrenergic neurons o f the guinea-pig vas deferens (Thoa et al., 1969), the dog saphenous veins and cerebral arteries (Verbeuren et al., 1983) and the rat mescnteric arteries (Kawasaki and Takasaki, 1984). An extraneuronal accumulation o f 5-HT has also been described in rat and rabbit lungs (Junod, 1972; Iwasawa and Gillis, 1974), rabbit ear artery (Buchan et al., 1974), dog saphenous veins (Paiva et al., 1984), rat aorta (Fukuda et al., 1986) and rat heart (Bryan et al., 1989).
*To whom all correspondence should be addressed. 613
The aim of the present investigation was to examine the uptake of 5-HT in rat isolated atria and its mechanism, especially the possibility of a storage in the adrenergic nerves when the tissue is incubated with a high concentration of 5-HT. MATERIALS AND METHODS
Rat isolated atria Male Sprague-Dawley rats (Charles River, France), weighing 250-350 g were killed by a blow on the head and exsanguinated. Atria were rapidly dissected out and set up in an organ bath which contained 3 ml of Krebs-Henseleit solution of the following composition (mmol/I): NaCI, 118; KCI, 4.7; CaCI 2, 2.5; MgSO4, 0.45; N a H C O , 25; KH2PO 4, 1; glucose, I1.1; Naz-EDTA, 0.07; ascorbic acid, 0.07; and 0.7 /amol/l atropine sulphate. The organ bath was kept at 37°C and was constantly gassed with 5% CO 2 in 02. A rest of 30 rain preceded any experiment to allow atria equilibration. Uptake of radioactive neurotransmitters After the atria equilibration period, ~'C-5-HT (24 kBq/ml of bath) or 3H-NA (37 kBq/ml of bath) was added to the organ bath and maintained for 20 rain. Non-labelled 5-HT or NA was also introduced at the same time as ~4C-5-HT or 3H-NA respectively to reach a final concentration of 46/a M for 5-HT and 0.4/~M for NA. An aliquot of the bath was collected at the beginning of the incubation time and weighed for later radioactivity counting (RE). At the end of the incubation time, atria were washed for 60 rain with the Krebs-Henseleit solution (7 ml/min), dried, weighed, pounded in 1 ml perchloric acid 0.4 M (4°C, 30 sec) and centrifuged (4000&, 4°C, 10 min). Supernatant containing the atria radioactivity was counted (R^). The ratio values (R) were calculated as: R = R^ (dpm per mg of tissue)/Ri (dpm per mg of bath). The atria incubated in the same
CHARLES EL RAWADIet al.
614
conditions as described above in the absence of any drug served as control. A control group was performed for each radioactive sample. The drugs were added to the bath 10 min before the incubation with labelled neurotransmitter except for reserpine and 6-hydroxydopamine (6-OHDA) which were injected to the rats. Reserpine (2.5 mg/kg) was administered i.p. 48 and 24 hr before experiment while 6-OHDA (I00 mg/kg) was injected i.p. 48 hr before experiment. The non-specific-binding values were determined in the same experimental conditions as for control, using for incubation of atria the same quantity of labelled neurotransmitter and a more important concentration of non labelled neurotransmitter: final concentration of 2500/~ M for 5-HT and 40,aM for NA.
hydroxytryptamine creatinine sulphate (Sigma), noradrenaline bitartrate (Sigma), hydrocortisone hemisuccinate (Roussel), 6-hydroxydopamine hydrobromide (Sigma), reserpine (Ciba-Geigy), desipramine hydrochloride (CibaGeigy), fluvoxamine maleate (Duphar), indalpine (Rh6nePoulenc Sant6), methiothepin maleate (Hoffmann-La Roche), MDL 72222 methane sulphonate (Merrell Dow Research Institute).
Statistical analysis Results are expressed as mean + SEM. Statistical analysis of the data was performed by analysis of variance followed by the Scheffe test for the comparisons between groups.
HPLC measurements 20~1 of the supernatant obtained as described above were injected into the HPLC system to detect NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents. The HPLC system consisted of a Waters model 510 solvent delivery system, Clin-Rep plasma catecholamine column protected by a guard column (Guard-PaL C18) and an electrochemical detector M 460, all obtained from Waters. The mobile phase (pH 4.3) contained 21 mM citric acid, 50mM sodium acetate, 0.107mM Na2-EDTA, 1.06mM dibutylamine and 2.8 mM octane sulfonic acid (Waters, Pic B8) to which 5% (vol/vol) methanol was added. The mobile phase was filtered (0.45 ~um filter, Millipore) and degassed under vacuum before use. A flow-rate of 0.9 ml/min was used.
Radioactivity counting The samples were added to 8ml scintillation fluid (Picofluor). Radioactivity was counted in a Packard Tricarb 4530 liquid scintillation counter.
Drugs 5-Hydroxy[ 1-'4 C]tryptamine ( i 5 x 10~k Bq/mmol; CEA), DL-[7,8-3H] noradrenaline (15 x 10SkBq/mmol; CEA), 5-
RESULTS The atria removed immediately by a blow on the head and briefly K r e b s - H e n s e l e i t solution contained o f 5 - H T / m g o f tissue (n = 7), while undetectable.
from rats killed washed with the 3.71 + 0.43 pmol the 5 - H I A A was
Uptake of t~C-S-HT After a 20 min incubation with IaC-5-HT (46/aM; 24 kBq/ml) (control group, n = 5), the radioactivity value (RA) was 198 + 1 5 d p m / m g o f atria and the ratio (R): radioactivity per mg o f tissue/radioactivity per mg o f organ bath was 0.18 + 0.014 (Table 1). The atria NA, 5-HT and 5 - H I A A contents measured by H P L C at the end o f the experiments were respectively 8.55 + 0.75, 5.91 + 0.37 and 2.59 + 0.25 p m o l / m g o f tissue. In the same conditions, a preincubation with h y d r o c o r t i s o n e (150/aM, n = 6 ) 10min before the '4C-5-HT addition was without any effect neither on RA and R nor on the NA, 5-HT and 5 - H I A A values.
Table 1. The effect of drugs on the uptake of *4C-5-HT by rat isolated atria and on the NA, 5-HT and 5-HIAA atria contents measured by HPLC after a 20 rain incubation with )4C-5-HT (46 ~M, 24 kBq/ml)
Control
~4C-5-HT uptake (dpm/mg) 198.+15
R 0.18 + 0.014
NA (pmol/mg) 8.55_+0.75
5-HT (pmol/mg) 5.91 __0.37
5-HIAA (pmol/mg) 2.59_+0.25
(n = 5)
Hydrocortisone (150~M) (n =6)
192_+11
0.17+0.011
9.72-+0.40
6.20-+0.65
2.44+0.19
Control
182-+9
0.13-+0.006
9.16_+1.30
4.94_+0.67
2.30+0.24
1,10__ 17t
0.10 __0.004t
4.73-+3.60"t"
3.23_+0.37
2.62 __0.41
123 _+5~:
0.09 .+ 0.004:~
0.40 _+0.06:~
2.33 _+0.3 Vr
2.62 _+0.36
66 _+6~
0.03 -+ 0.003~: 7.97_+0.44
4.97_+0.58
1.62_+0.22
5.45 + 0.63
2.08 ___0.21
(n = 7)
6-OHDA (100 m g / k g ) (n = 4)
Reserpine (2.5 mg/kg) (n = 5) Non-specific-binding (n = 6) Control
160_+ 16
0.14+0.017
147 _+ 15
0.14 _+ 0.013
165-+13
0.16-+0.011
8.25-+0.76
7.30_+2.11
2.17_+0.17
215.+14
0.18-+0.012
9.68_+1.24
6.83.+0.76
3.06_+0.47
180_+ 14
0.15_+0.013" 11.10_+0.73
4.98+0.38
3.46_+0.32
(n = 5)
Desipramine
(1 ,aM) (n = 7) Methiotbepin (0.75 ~uM) + MDL 72222 (0.75 ~M) (n = 7) Control
10.35 _+ 0.63
(n = 5)
Fluvoxamine ( 1 / ~ M ) (n = 6)
Indalpine 143 _+8t 0.12 _+0.006t 10.06 _+0.66 4.49 _+0.53* 3.52 .+ 0.34 (5,aM) (n = 5) R = ratio, radioactivity per mg of tissue at the end of the experiment/radioactivityper mg of organ bath collected at the beginning of the incubation. Significant difference*P < 0.05, tP < 0.01, ~:P < 0.001 vs corresponding controls.
Uptake of 5-HT in rat isolated atria The treatment of rats with 6 - O H D A (n = 4 ) decreased the radioactivity value (RA) from 182_+ 9 d p m / m g in control group (n = 7) to 140 -+ 17 d p m / mg (P < 0.01) and the R value from 0.13 _+ 0.006 in control group to 0.10 -+ 0.004 (P < 0.01). Also, the N A content was reduced from 9.16 ± 1.30 p m o l / m g to 4.73 -+ 3 . 6 0 p m o l / m g (P <0.01) while the 5-HT and 5 - H I A A contents were unchanged. The treatment of rats with reserpine (n = 5) decreased the RA tO 123-+ 5 d p m / m g (P <0.001), the R value to 0.09-+0.004 ( P < 0 . 0 0 1 ) , the N A content to 0.40 -+ 0.06 pmol/mg (P < 0.001), the 5-HT content to 2.33 ±0.31 (P < 0.01) while the 5 - H I A A content remained unchanged. In the conditions of nonspecific-binding measurement for ~4C-5-HT (n = 6), the radioactivity measured was 6 6 - + 6 d p m / m g (P < 0.001) and the R value 0.03 -+ 0.003 which is 23% of the control group value. The preincubation of atria with desipramine ( l # M , n = 7 ) or with methiothepin (0.75/~M)+ M D L 72222 (0.75/~M) (n = 7 ) 10rain before the ~4C-5-HT addition was without any effect on the results compared to control group (n = 5). In contrast, the preincubation with fluvoxamine (1/~M, n = 6 ) reduced the R value from 0 . 1 8 + 0 . 0 1 2 in control group ( n = 5 ) to 0.15-+0.013 ( P < 0 . 0 5 ) while the other data were unchanged (RA, N A , 5-HT and 5-HIAA). On the other hand, the preincubation with indalpine (5/~M, n = 5) decreased the radioactivity value from 215 _+14 d p m / m g in control group to 143 -+ 8 d p m / m g (P < 0.01), the R value from 0.18_+0.012 to 0.12_+0.006 ( P < 0 . 0 1 ) , the 5-HT content from 6.83 -+ 0.76 pmol/mg to 4.49 -+ 0.53 p m o l / m g (P < 0.05) while the N A and 5 - H I A A contents were unchanged.
measured by H P L C at the end of the experiments were 26.30 -+ 1.44 and 3.74_+ 0.66 pmol/mg respectively. N o 5 - H I A A was detected. After reserpine treatment (n = 6), the uptake of N A was reduced to 1908 +_ 307 d p m / m g (P < 0.001 vs control group) and the R value to 0.62_+0.10 (P < 0.001). N o N A was detected by H P L C while the 5-HT value was unchanged. The 5 - H I A A content was 0.18 _ 0.02 pmol/mg. The presence of 5-HT ( 5 0 # M , n = 6 ) in the organ bath at the same time as 3 H - N A reduced the N A uptake to 7566 _+ 207 d p m / m g (P < 0.001) and the R value to 2 . 5 3 _ 0 . 1 2 ( P < 0 . 0 0 1 ) . The N A content measured by H P L C was also decreased to 18.62_+ 4.43 pmol/mg (P < 0.01), while the 5-HT and 5 - H I A A contents were increased to 6.75_+ 1.58 pmol/mg (P < 0.01) and 1.78 _ 0.36 pmol/mg (P <0.001) respectively. In the group of 3 H - N A non-specific-binding measurement, the radioactivity value was 471 - + 9 7 d p m / m g (P <0.001) and the R value 0.24 _4-0.05 which is 5% of the control value. In a second group of experiments, a 10min atria preincubation with desipramine ( l / z M , n = 6) before the 3H-NA addition reduced the N A uptake from 7282 + 793 d p m / m g (control group, n = 6) to 830 _+ 58 d p m / m g (P < 0.001) and the R value from 4.12_+0.30 to 0.49-+0.03 ( P < 0 . 0 0 1 ) , while the NA, 5-HT and 5 - H I A A contents were unchanged. In contrast, the presence of fluvoxamine ( l / ~ M , n = 6) in the organ bath l0 min before the 3H-NA addition was without any effect neither on the N A uptake and on the R value, nor on the atria NA, 5-HT and 5 - H I A A contents compared to the control group (n = 6).
Uptake of 3H-NA The radioactivity taken up in atria (R^) after a 20min incubation with 3 H - N A (0.4/~M; 37 kBq/ml) (control group, n = 6) was 12870-+ 615 d p m / m g of tissue (Table 2). The ratio (R): radioactivity per mg of tissue/radioactivity per mg of bathing solution was 4.40 +_ 0.18. The N A and 5-HT atria contents
615
DISCUSSION The present study was designed to investigate whether the rat isolated atria could take up the 5-HT and to compare the characteristics of 5-HT uptake and N A uptake. For this aim, we incubated the preparations, as described in Methods, with either
Table 2. The effect of drugs on the uptake of 3H-NA by rat isolated atria and on the NA, 5-HT and 5-HIAA atria contents measured by HPLC after a 20 rain incubation with 3H-NA (0.4/aM, 37 kBq/ml) 3H-NA uptake NA 5-HT 5-HIAA (dpm/mg) R (pmol/mg) ( p m o l / m g ) (pmol/mg) Control 12870 + 615 4.40+0.18 26.304_ 1.44 3.744_0.66
Reserpine (2.5 mg/kg) (n = 6) 5-HT (50/aM)
1908 -+ 307++
0.62 4_0.10++
7566+207++
2.53_+0.12++
< D.L.
2.95 _+0.28
0.18 4_0.02
18.62 -.- 4.431.
6.754-1.58t"
1.78 + 0.36~
4.124-0.30
10.66_+0.39
2.444-0.19
0.49 4_ 0.03.+
11.57 4- 1.04
2.62 _+ 0.40
< D.L.
10153+275
5.634-0.26
10.184-0.52
3.344_0.47
9375_+482
5.58_+0.33
12.11 +_1.05
3.184_0.26
(n = 6)
Non-speofic-binding
471 _+.97~
9.24 ± 0.05~
(n = 6)
Control
72824-793
(n = 6)
Dcsipramine
(I juM) (n = 6) Control
830 _ 58++
(n = 6)
Fluvoxamine ( 1 / ~ M ) (n = 6)
R - ratio, radioactivity per mg of tissue at the end of the experiment/radioactivityper mg of organ bath collected at the beginning of the incubation. D.L. = detection limits of the HPLC method. Significant differencetP < 0.01, ++P< 0.001 vs corresponding controls.
616
CHARLES EL RAWADIel al.
'4C-5-HT or 3H-NA and we measured by radioactivity counting the uptake of the labelled neurotransmitters and by HPLC the total atria contents in NA, 5-HT and 5-HIAA: the main metabolite of 5-HT. In a first step, we demonstrated that 5-HT like NA are taken into the rat atria since the fixed radioactivity after the incubation with either ~4C-5-HT or 3H-NA is more important than the non-specific binding values. This radioactivity fixed by atria is not an interaction with specific receptors. The washing procedure of 1 hr applicated after the incubation period makes easier the removal from its receptors any eventual labelled neurotransmitter. We confirmed this hypothesis in the case of ~4C-5-HT since the preincubation of atria with methiothepin + MDL 72222, which antagonise the most known 5-HT receptors, did not modify the value of ~*C-5-HT fixed. Two modes of 5-HT uptake are described in literature: an intraneuronal uptake into the noradrenergic neurons (Thoa et al., 1969; Verbeuren et al., 1983; Kawasaki and Takasaki, 1984) and an extraneuronal accumulation of a still unclear mechanism (Junod, 1972; Iwasawa and Gillis, 1974; Buchan et al., 1974; Paiva et al., 1984; Fukuda et al., 1986; Bryan et al., 1989). In this study, we were especially interested by the 5-HT intraneuronal uptake hypothesis. Our results showed that the destruction of sympathetic nerves within the atria using 6-OHDA resulted in a decrease in ~4C-5-HT uptake. The destruction of the nerves is confirmed by the lower NA levels measured by HPLC compared to controls. Also, the treatment of rats with reserpine which inhibits the uptake of NA into its storage vesicles within the sympathetic terminals reduced the ~4C-5-HT uptake into the atria. To valid our method of reserpine treatment, we studied the 3H-NA uptake into the atria obtained from reserpine treated rats in the same conditions as for ~4C-5-HT experiments. The results showed a strong decrease in 3H-NA uptake and a total NA depletion measured by HPLC. We conclude that 5-HT is taken into the NA storage vesicles in the sympathetic nerves. We noticed that a competition exists between NA and 5-HT uptake at the concentrations used since the incubation of atria with 5-HT decreased the uptake of 3H.NA. We examined the hypothesis that the two neurotransmitters could use the same carrier at the sympathetic neuronal membranes. For this aim, we measured the effect of desipramine, a specific antagonist of the NA carrier (Claassen, 1983) and the effect of fluvoxamine and indalpine, two specific antagonists of the 5-HT carrier at the serotonergic terminals (Claassen, 1983; Le Fur and Uzan, 1977) on the uptake of ~4C-5-HT and 3H-NA. Desipramine decreased the N A uptake while fluvoxamine had no effect on it. This confirms the selectivity of these two drugs. On the other hand, desipramine had no effect on 5-HT uptake and the decrease of this uptake was not very important with fluvoxamine and indalpine. Thus, the uptake of 5-HT did not use the NA carrier and the 5-HT carrier is not the main way of 5-HT transport into noradrenergic nerves. After reserpine, a quantity of neurotransmitters is still taken up by atria since the radioactivity counting did not reach the values of the non-specific binding after this
treatment. This quantity may represent the extraneuronal uptake. The mechanism of this uptake is more clear in the case of NA than for 5-HT: it is called uptake2 compared to the uptake] which is intraneuronal and inhibited by reserpine. The uptake2 is mediated via an energy-dependent transport mechanism which is probably localized in the membranes of the extraneuronal ceils (Nedergaard, 1988) and is inhibited by corticosteroids (Iversen and Salt, 1970). The mechanism of the 5-HT extraneuronal accumulation is not the same as for NA since hydrocortisone did not decrease the ~C-5-HT uptake. This accumulation may be associated with the presence of granules within the atrial muscle cells (Theron et al., 1978). A part of this extraneuronal uptake is intracellular and metabolized by the MAO since a stable amount of 5-HIAA appeared whenever we incubated the atria with exogenous 5-HT and was not reduced by reserpine and 6-OHDA. When no exogenous 5-HT was added, the atria 5-HT levels measured by HPLC were unchanged in all our experimental groups. These results were close to those we found in atria immediately after the rats sacrifice in the absence of any treatment. This 5-HT may be located into residual platelets that the preparations can contain. In conclusion, 5-HT is taken into the storage vesicles of the rat atrial noradrenergic nerves. The way used to cross the nerve membranes does not implicate the classic NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation of 5-HT of an unknown mechanism was also noticed. A part of this extraneuronal accumulation is intracellular. REFERENCES
Allen G. S., Henderson L. M., Chou S. N. and French L. A. (1974) Cerebral arterial spasm. Part 2: in vitro contractile activity of serotonin in human serum and CSF on the canine basilar artery, and its blockade by methysergide and phenoxybenzamine. J. Neurosurg. 40, 442-450. Angus J. A., Brazenor R. M. and Le Duc M. A. (1982) Verapamih a selective antagonist of constrictor substances in coronary artery: implication for variant angina. Clin. exp. Pharmac. Physiol. 6, 15-28. Bove A. A. and Dewey J. D. (1983) Effects of serotonin and histamine on proximal and distal coronary vasculature in dogs: comparison with alpha-adrenergic stimulation. Am. J. Cardiol. 52, 1333-1339. Bryan L. J., O'Donnell S. R. and Williams A. M. (1989) Dissipation mechanisms for 5-hydroxytryptamine in the coronary circulation of the isolated perfused heart of the rat. Br. J. Pharmac. 97, 329-338. Buchan P., Lewis A. J. and Sugrue M. F. (1974) A comparison of the accumulation of noradrenaline and 5-hydroxytryptamine into arterial smooth muscle. Br. J. Pharmac. 52, 132P-133P. Claasen V. (1983) Review of the animal pharmacology and pharmacokinetics of fluvoxamine. Br. J. clin. Pharmac. 15, 349S-355S. Fukuda S., Su C. and Lee T. J.-F. (1986) Mechanisms of extraneuronal serotonin uptake in the rat aorta. J. Pharmac. exp. Ther. 239, 264-269. Halpern A., Kuhn P. H., Shaftel H. E., Samuels S. S. and Shaftel N. (1960) Raynaud's disease, Raynaud's phenomenon, and serotonin. Angiology 11, 151-167. Iversen L. L. and Salt P. J. (1970) Inhibition of catecholamine uptake2 by steroids in the isolated rat heart. Br. J. Pharmac. 40, 528-530.
Uptake of 5-HT in rat isolated atria Iwasawa Y. and Gillis C. N. (1974) Pharmacological analysis of norepinephrine and 5-hydroxytryptamine removal from the pulmonary circulation: differentiation of uptake sites of each amine. J. Pharmac. exp. Ther. 188, 386-393. Junod A. F. (1972) Uptake, metabolism and efltux of 14C-5-hydroxytryptamine in isolated perfused rat lungs. J. Pharmac exp. Ther. 183, 341-355. Kawasaki H. and Takasaki K. (1984) Vasoconstrictor response induced by 5-hydroxytryptamin¢ released from vascular adrenergic nerves by periarterial nerve stimulation. J. Pharmac. exp. Ther. 229, 816-822. Le Fur G. and Uzan A. (1977) Effects of 4-(3-indolyl-alkyl) piperidine derivatives on uptake and release of noradrenaline, dopamine and 5-hydroxytryptamine in rat brain synaptosomes, rat heart and human blood platelets. Biochem. Pharmac. 26, 497-503. Nedergaard O. A. (1988) Catecholamines: regulation, re!ease and inactivation. Pharmac. Toxic. 63 (Suppl. I), 5-8. Paiva M. Q., Caramona M. and Osswald W. (1984) Intraand extraneuronal metabolism of 5-hydroxytryptamine in the isolated saphenous vein of the dog. NaunynSchmiedeberg's Arch. Pharmac. 325, 62-68. Raskin N. H. (1981) Pharmacology of migraine. A. Rev. Pharmac. Toxic. 21, 463-478.
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Shepro D., Batbouta J. C., Robblee L. S., Carson M. P. and Belamarich F. A. (1975) Serotonin transport by cultured bovine aortic endothelium. Circulation Res. 36, 799-806. Stahl S. M. and Meltzer H. Y. (1978) A kinetic and pharmacological analysis of 5-hydroxytryptamine transport by human ptatelets and platelet storage granules: comparison with central serotonergic neurons. J. Pharmac exp. Ther. 205, 118-132. Stranden E., Roald O. K. and Krohg K. (1982) Treatment of Raynaud's phenomenon with the 5-HT2-receptor antagonist ketanserin. Br. med. J. 285, 1069-1071. Theron J. J., Biagio R., Meyer A. C., Boekkooi S. and Seegers J. C. (1978) The effect of a serotonin inhibitor on the serotonin content and ultra-structure of rat atria and ventricles with special reference to atrial granules. Life Sci. 23, 111-120. Thoa N. B., Eccleston D. and Axelrod J. (1969) The accumulation of ~4C-serotonin in the guinea-pig vas deferens. J. Pharmac. exp. Ther. 169, 68-73. Verbeuren T. J., Jordaens F. H. and Herman A. G. (1983) Accumulation and release of [3H]-5-hydroxytryptamine in saphenous veins and cerebral arteries of the dog. J. Pharmac. exp. Ther. 226, 579-588.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
UPTAKE OF 5 - H Y D R O X Y T R Y P T A M I N E IN RAT ISOLATED ATRIA CHARLES EL RAWADI,* MICHI~LE HEIMBURGER, MONIQUE DAVY, MICHI~LEMIDOL-MONNET, FRANqOlSE BESLOTand YvES COHEN Laboratoire de Pharmacologie, Facult~ de Pharmacie, F-92296 Ch~tenay-Malabry, France [Fax 4683-1303] (Received 14 January 1992) AI~--I. The mode of 5-hydroxytryptamine (5-HT) uptake by the rat isolated atria was studied and compared to noradrenaline (NA) uptake. 2. Rat isolated atria were incubated with ~'C-5-HT (46/aM) or 3H-NA (0.4/aM). After washing, the radioactivity fixed in atria was counted and the total NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents were measured by HPLC. 3. ~4C-5-HT uptake was reduced in atria from 6-hydroxydopamine (100mg/kg, i.p., 48hr before experiments) or reserpine (2.5 mg/kg, i.p., 24 and 48 hr before experiments) pretreated rats. 4. The incubation of atria with 5-HT (50/aM) at the same time as 3H-NA reduced the 3H-NA value fixed. 5. Addition of desipramine (I/aM) or hydrocortisone (150/aM) before the incubation of atria with ~4C-5-HT was without any effect on ~4C-5-HT uptake. In contrast, fluvoxamine (1/aM) or indalpine (5/aM) caused a slight inhibition. 6. These data indicate that 5-HT is taken into the NA storage vesicles within the atria sympathetic nerves. This uptake does not use the NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation was noticed and a part of it is intracellular.
INTRODUCTION In physiological conditions, there is no circulating 5-hydroxytryptamine (5-HT) because it is removed by platelets or vascular endothelium (Stahl and Meltzer, 1978; Shepro et al., 1975). In various pathological conditions, an excess of 5-HT is released from platelets and saturates the uptake by nonaggregating platelets and endothelium. These conditions are observed in the coronary vasospasm of Prinzmetars angina (Angus et al., 1982; Bove and Dewey, 1983), cerebral vasospasm after subarachnoid haemorrhage (Allen et al., 1974), digital vasospasm o f R a y n a u d ' s phenomenon (Halpern et al., 1960; Stranden et al., 1982) and migraine (Raskin,1981). The excess of 5-HT may then reach the heart, the vascular smooth muscle cells and/or sympathetic nerve terminals to elicit its different responses. The uptake of 5-HT out of serotonergic structures and the mechanism of this process were examined by different authors in various preparations. 5-HT is taken up and stored in noradrenergic neurons o f the guinea-pig vas deferens (Thoa et al., 1969), the dog saphenous veins and cerebral arteries (Verbeuren et al., 1983) and the rat mescnteric arteries (Kawasaki and Takasaki, 1984). An extraneuronal accumulation o f 5-HT has also been described in rat and rabbit lungs (Junod, 1972; Iwasawa and Gillis, 1974), rabbit ear artery (Buchan et al., 1974), dog saphenous veins (Paiva et al., 1984), rat aorta (Fukuda et al., 1986) and rat heart (Bryan et al., 1989).
*To whom all correspondence should be addressed. 613
The aim of the present investigation was to examine the uptake of 5-HT in rat isolated atria and its mechanism, especially the possibility of a storage in the adrenergic nerves when the tissue is incubated with a high concentration of 5-HT. MATERIALS AND METHODS
Rat isolated atria Male Sprague-Dawley rats (Charles River, France), weighing 250-350 g were killed by a blow on the head and exsanguinated. Atria were rapidly dissected out and set up in an organ bath which contained 3 ml of Krebs-Henseleit solution of the following composition (mmol/I): NaCI, 118; KCI, 4.7; CaCI 2, 2.5; MgSO4, 0.45; N a H C O , 25; KH2PO 4, 1; glucose, I1.1; Naz-EDTA, 0.07; ascorbic acid, 0.07; and 0.7 /amol/l atropine sulphate. The organ bath was kept at 37°C and was constantly gassed with 5% CO 2 in 02. A rest of 30 rain preceded any experiment to allow atria equilibration. Uptake of radioactive neurotransmitters After the atria equilibration period, ~'C-5-HT (24 kBq/ml of bath) or 3H-NA (37 kBq/ml of bath) was added to the organ bath and maintained for 20 rain. Non-labelled 5-HT or NA was also introduced at the same time as ~4C-5-HT or 3H-NA respectively to reach a final concentration of 46/a M for 5-HT and 0.4/~M for NA. An aliquot of the bath was collected at the beginning of the incubation time and weighed for later radioactivity counting (RE). At the end of the incubation time, atria were washed for 60 rain with the Krebs-Henseleit solution (7 ml/min), dried, weighed, pounded in 1 ml perchloric acid 0.4 M (4°C, 30 sec) and centrifuged (4000&, 4°C, 10 min). Supernatant containing the atria radioactivity was counted (R^). The ratio values (R) were calculated as: R = R^ (dpm per mg of tissue)/Ri (dpm per mg of bath). The atria incubated in the same
CHARLES EL RAWADIet al.
614
conditions as described above in the absence of any drug served as control. A control group was performed for each radioactive sample. The drugs were added to the bath 10 min before the incubation with labelled neurotransmitter except for reserpine and 6-hydroxydopamine (6-OHDA) which were injected to the rats. Reserpine (2.5 mg/kg) was administered i.p. 48 and 24 hr before experiment while 6-OHDA (I00 mg/kg) was injected i.p. 48 hr before experiment. The non-specific-binding values were determined in the same experimental conditions as for control, using for incubation of atria the same quantity of labelled neurotransmitter and a more important concentration of non labelled neurotransmitter: final concentration of 2500/~ M for 5-HT and 40,aM for NA.
hydroxytryptamine creatinine sulphate (Sigma), noradrenaline bitartrate (Sigma), hydrocortisone hemisuccinate (Roussel), 6-hydroxydopamine hydrobromide (Sigma), reserpine (Ciba-Geigy), desipramine hydrochloride (CibaGeigy), fluvoxamine maleate (Duphar), indalpine (Rh6nePoulenc Sant6), methiothepin maleate (Hoffmann-La Roche), MDL 72222 methane sulphonate (Merrell Dow Research Institute).
Statistical analysis Results are expressed as mean + SEM. Statistical analysis of the data was performed by analysis of variance followed by the Scheffe test for the comparisons between groups.
HPLC measurements 20~1 of the supernatant obtained as described above were injected into the HPLC system to detect NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents. The HPLC system consisted of a Waters model 510 solvent delivery system, Clin-Rep plasma catecholamine column protected by a guard column (Guard-PaL C18) and an electrochemical detector M 460, all obtained from Waters. The mobile phase (pH 4.3) contained 21 mM citric acid, 50mM sodium acetate, 0.107mM Na2-EDTA, 1.06mM dibutylamine and 2.8 mM octane sulfonic acid (Waters, Pic B8) to which 5% (vol/vol) methanol was added. The mobile phase was filtered (0.45 ~um filter, Millipore) and degassed under vacuum before use. A flow-rate of 0.9 ml/min was used.
Radioactivity counting The samples were added to 8ml scintillation fluid (Picofluor). Radioactivity was counted in a Packard Tricarb 4530 liquid scintillation counter.
Drugs 5-Hydroxy[ 1-'4 C]tryptamine ( i 5 x 10~k Bq/mmol; CEA), DL-[7,8-3H] noradrenaline (15 x 10SkBq/mmol; CEA), 5-
RESULTS The atria removed immediately by a blow on the head and briefly K r e b s - H e n s e l e i t solution contained o f 5 - H T / m g o f tissue (n = 7), while undetectable.
from rats killed washed with the 3.71 + 0.43 pmol the 5 - H I A A was
Uptake of t~C-S-HT After a 20 min incubation with IaC-5-HT (46/aM; 24 kBq/ml) (control group, n = 5), the radioactivity value (RA) was 198 + 1 5 d p m / m g o f atria and the ratio (R): radioactivity per mg o f tissue/radioactivity per mg o f organ bath was 0.18 + 0.014 (Table 1). The atria NA, 5-HT and 5 - H I A A contents measured by H P L C at the end o f the experiments were respectively 8.55 + 0.75, 5.91 + 0.37 and 2.59 + 0.25 p m o l / m g o f tissue. In the same conditions, a preincubation with h y d r o c o r t i s o n e (150/aM, n = 6 ) 10min before the '4C-5-HT addition was without any effect neither on RA and R nor on the NA, 5-HT and 5 - H I A A values.
Table 1. The effect of drugs on the uptake of *4C-5-HT by rat isolated atria and on the NA, 5-HT and 5-HIAA atria contents measured by HPLC after a 20 rain incubation with )4C-5-HT (46 ~M, 24 kBq/ml)
Control
~4C-5-HT uptake (dpm/mg) 198.+15
R 0.18 + 0.014
NA (pmol/mg) 8.55_+0.75
5-HT (pmol/mg) 5.91 __0.37
5-HIAA (pmol/mg) 2.59_+0.25
(n = 5)
Hydrocortisone (150~M) (n =6)
192_+11
0.17+0.011
9.72-+0.40
6.20-+0.65
2.44+0.19
Control
182-+9
0.13-+0.006
9.16_+1.30
4.94_+0.67
2.30+0.24
1,10__ 17t
0.10 __0.004t
4.73-+3.60"t"
3.23_+0.37
2.62 __0.41
123 _+5~:
0.09 .+ 0.004:~
0.40 _+0.06:~
2.33 _+0.3 Vr
2.62 _+0.36
66 _+6~
0.03 -+ 0.003~: 7.97_+0.44
4.97_+0.58
1.62_+0.22
5.45 + 0.63
2.08 ___0.21
(n = 7)
6-OHDA (100 m g / k g ) (n = 4)
Reserpine (2.5 mg/kg) (n = 5) Non-specific-binding (n = 6) Control
160_+ 16
0.14+0.017
147 _+ 15
0.14 _+ 0.013
165-+13
0.16-+0.011
8.25-+0.76
7.30_+2.11
2.17_+0.17
215.+14
0.18-+0.012
9.68_+1.24
6.83.+0.76
3.06_+0.47
180_+ 14
0.15_+0.013" 11.10_+0.73
4.98+0.38
3.46_+0.32
(n = 5)
Desipramine
(1 ,aM) (n = 7) Methiotbepin (0.75 ~uM) + MDL 72222 (0.75 ~M) (n = 7) Control
10.35 _+ 0.63
(n = 5)
Fluvoxamine ( 1 / ~ M ) (n = 6)
Indalpine 143 _+8t 0.12 _+0.006t 10.06 _+0.66 4.49 _+0.53* 3.52 .+ 0.34 (5,aM) (n = 5) R = ratio, radioactivity per mg of tissue at the end of the experiment/radioactivityper mg of organ bath collected at the beginning of the incubation. Significant difference*P < 0.05, tP < 0.01, ~:P < 0.001 vs corresponding controls.
Uptake of 5-HT in rat isolated atria The treatment of rats with 6 - O H D A (n = 4 ) decreased the radioactivity value (RA) from 182_+ 9 d p m / m g in control group (n = 7) to 140 -+ 17 d p m / mg (P < 0.01) and the R value from 0.13 _+ 0.006 in control group to 0.10 -+ 0.004 (P < 0.01). Also, the N A content was reduced from 9.16 ± 1.30 p m o l / m g to 4.73 -+ 3 . 6 0 p m o l / m g (P <0.01) while the 5-HT and 5 - H I A A contents were unchanged. The treatment of rats with reserpine (n = 5) decreased the RA tO 123-+ 5 d p m / m g (P <0.001), the R value to 0.09-+0.004 ( P < 0 . 0 0 1 ) , the N A content to 0.40 -+ 0.06 pmol/mg (P < 0.001), the 5-HT content to 2.33 ±0.31 (P < 0.01) while the 5 - H I A A content remained unchanged. In the conditions of nonspecific-binding measurement for ~4C-5-HT (n = 6), the radioactivity measured was 6 6 - + 6 d p m / m g (P < 0.001) and the R value 0.03 -+ 0.003 which is 23% of the control group value. The preincubation of atria with desipramine ( l # M , n = 7 ) or with methiothepin (0.75/~M)+ M D L 72222 (0.75/~M) (n = 7 ) 10rain before the ~4C-5-HT addition was without any effect on the results compared to control group (n = 5). In contrast, the preincubation with fluvoxamine (1/~M, n = 6 ) reduced the R value from 0 . 1 8 + 0 . 0 1 2 in control group ( n = 5 ) to 0.15-+0.013 ( P < 0 . 0 5 ) while the other data were unchanged (RA, N A , 5-HT and 5-HIAA). On the other hand, the preincubation with indalpine (5/~M, n = 5) decreased the radioactivity value from 215 _+14 d p m / m g in control group to 143 -+ 8 d p m / m g (P < 0.01), the R value from 0.18_+0.012 to 0.12_+0.006 ( P < 0 . 0 1 ) , the 5-HT content from 6.83 -+ 0.76 pmol/mg to 4.49 -+ 0.53 p m o l / m g (P < 0.05) while the N A and 5 - H I A A contents were unchanged.
measured by H P L C at the end of the experiments were 26.30 -+ 1.44 and 3.74_+ 0.66 pmol/mg respectively. N o 5 - H I A A was detected. After reserpine treatment (n = 6), the uptake of N A was reduced to 1908 +_ 307 d p m / m g (P < 0.001 vs control group) and the R value to 0.62_+0.10 (P < 0.001). N o N A was detected by H P L C while the 5-HT value was unchanged. The 5 - H I A A content was 0.18 _ 0.02 pmol/mg. The presence of 5-HT ( 5 0 # M , n = 6 ) in the organ bath at the same time as 3 H - N A reduced the N A uptake to 7566 _+ 207 d p m / m g (P < 0.001) and the R value to 2 . 5 3 _ 0 . 1 2 ( P < 0 . 0 0 1 ) . The N A content measured by H P L C was also decreased to 18.62_+ 4.43 pmol/mg (P < 0.01), while the 5-HT and 5 - H I A A contents were increased to 6.75_+ 1.58 pmol/mg (P < 0.01) and 1.78 _ 0.36 pmol/mg (P <0.001) respectively. In the group of 3 H - N A non-specific-binding measurement, the radioactivity value was 471 - + 9 7 d p m / m g (P <0.001) and the R value 0.24 _4-0.05 which is 5% of the control value. In a second group of experiments, a 10min atria preincubation with desipramine ( l / z M , n = 6) before the 3H-NA addition reduced the N A uptake from 7282 + 793 d p m / m g (control group, n = 6) to 830 _+ 58 d p m / m g (P < 0.001) and the R value from 4.12_+0.30 to 0.49-+0.03 ( P < 0 . 0 0 1 ) , while the NA, 5-HT and 5 - H I A A contents were unchanged. In contrast, the presence of fluvoxamine ( l / ~ M , n = 6) in the organ bath l0 min before the 3H-NA addition was without any effect neither on the N A uptake and on the R value, nor on the atria NA, 5-HT and 5 - H I A A contents compared to the control group (n = 6).
Uptake of 3H-NA The radioactivity taken up in atria (R^) after a 20min incubation with 3 H - N A (0.4/~M; 37 kBq/ml) (control group, n = 6) was 12870-+ 615 d p m / m g of tissue (Table 2). The ratio (R): radioactivity per mg of tissue/radioactivity per mg of bathing solution was 4.40 +_ 0.18. The N A and 5-HT atria contents
615
DISCUSSION The present study was designed to investigate whether the rat isolated atria could take up the 5-HT and to compare the characteristics of 5-HT uptake and N A uptake. For this aim, we incubated the preparations, as described in Methods, with either
Table 2. The effect of drugs on the uptake of 3H-NA by rat isolated atria and on the NA, 5-HT and 5-HIAA atria contents measured by HPLC after a 20 rain incubation with 3H-NA (0.4/aM, 37 kBq/ml) 3H-NA uptake NA 5-HT 5-HIAA (dpm/mg) R (pmol/mg) ( p m o l / m g ) (pmol/mg) Control 12870 + 615 4.40+0.18 26.304_ 1.44 3.744_0.66
Reserpine (2.5 mg/kg) (n = 6) 5-HT (50/aM)
1908 -+ 307++
0.62 4_0.10++
7566+207++
2.53_+0.12++
< D.L.
2.95 _+0.28
0.18 4_0.02
18.62 -.- 4.431.
6.754-1.58t"
1.78 + 0.36~
4.124-0.30
10.66_+0.39
2.444-0.19
0.49 4_ 0.03.+
11.57 4- 1.04
2.62 _+ 0.40
< D.L.
10153+275
5.634-0.26
10.184-0.52
3.344_0.47
9375_+482
5.58_+0.33
12.11 +_1.05
3.184_0.26
(n = 6)
Non-speofic-binding
471 _+.97~
9.24 ± 0.05~
(n = 6)
Control
72824-793
(n = 6)
Dcsipramine
(I juM) (n = 6) Control
830 _ 58++
(n = 6)
Fluvoxamine ( 1 / ~ M ) (n = 6)
R - ratio, radioactivity per mg of tissue at the end of the experiment/radioactivityper mg of organ bath collected at the beginning of the incubation. D.L. = detection limits of the HPLC method. Significant differencetP < 0.01, ++P< 0.001 vs corresponding controls.
616
CHARLES EL RAWADIel al.
'4C-5-HT or 3H-NA and we measured by radioactivity counting the uptake of the labelled neurotransmitters and by HPLC the total atria contents in NA, 5-HT and 5-HIAA: the main metabolite of 5-HT. In a first step, we demonstrated that 5-HT like NA are taken into the rat atria since the fixed radioactivity after the incubation with either ~4C-5-HT or 3H-NA is more important than the non-specific binding values. This radioactivity fixed by atria is not an interaction with specific receptors. The washing procedure of 1 hr applicated after the incubation period makes easier the removal from its receptors any eventual labelled neurotransmitter. We confirmed this hypothesis in the case of ~4C-5-HT since the preincubation of atria with methiothepin + MDL 72222, which antagonise the most known 5-HT receptors, did not modify the value of ~*C-5-HT fixed. Two modes of 5-HT uptake are described in literature: an intraneuronal uptake into the noradrenergic neurons (Thoa et al., 1969; Verbeuren et al., 1983; Kawasaki and Takasaki, 1984) and an extraneuronal accumulation of a still unclear mechanism (Junod, 1972; Iwasawa and Gillis, 1974; Buchan et al., 1974; Paiva et al., 1984; Fukuda et al., 1986; Bryan et al., 1989). In this study, we were especially interested by the 5-HT intraneuronal uptake hypothesis. Our results showed that the destruction of sympathetic nerves within the atria using 6-OHDA resulted in a decrease in ~4C-5-HT uptake. The destruction of the nerves is confirmed by the lower NA levels measured by HPLC compared to controls. Also, the treatment of rats with reserpine which inhibits the uptake of NA into its storage vesicles within the sympathetic terminals reduced the ~4C-5-HT uptake into the atria. To valid our method of reserpine treatment, we studied the 3H-NA uptake into the atria obtained from reserpine treated rats in the same conditions as for ~4C-5-HT experiments. The results showed a strong decrease in 3H-NA uptake and a total NA depletion measured by HPLC. We conclude that 5-HT is taken into the NA storage vesicles in the sympathetic nerves. We noticed that a competition exists between NA and 5-HT uptake at the concentrations used since the incubation of atria with 5-HT decreased the uptake of 3H.NA. We examined the hypothesis that the two neurotransmitters could use the same carrier at the sympathetic neuronal membranes. For this aim, we measured the effect of desipramine, a specific antagonist of the NA carrier (Claassen, 1983) and the effect of fluvoxamine and indalpine, two specific antagonists of the 5-HT carrier at the serotonergic terminals (Claassen, 1983; Le Fur and Uzan, 1977) on the uptake of ~4C-5-HT and 3H-NA. Desipramine decreased the N A uptake while fluvoxamine had no effect on it. This confirms the selectivity of these two drugs. On the other hand, desipramine had no effect on 5-HT uptake and the decrease of this uptake was not very important with fluvoxamine and indalpine. Thus, the uptake of 5-HT did not use the NA carrier and the 5-HT carrier is not the main way of 5-HT transport into noradrenergic nerves. After reserpine, a quantity of neurotransmitters is still taken up by atria since the radioactivity counting did not reach the values of the non-specific binding after this
treatment. This quantity may represent the extraneuronal uptake. The mechanism of this uptake is more clear in the case of NA than for 5-HT: it is called uptake2 compared to the uptake] which is intraneuronal and inhibited by reserpine. The uptake2 is mediated via an energy-dependent transport mechanism which is probably localized in the membranes of the extraneuronal ceils (Nedergaard, 1988) and is inhibited by corticosteroids (Iversen and Salt, 1970). The mechanism of the 5-HT extraneuronal accumulation is not the same as for NA since hydrocortisone did not decrease the ~C-5-HT uptake. This accumulation may be associated with the presence of granules within the atrial muscle cells (Theron et al., 1978). A part of this extraneuronal uptake is intracellular and metabolized by the MAO since a stable amount of 5-HIAA appeared whenever we incubated the atria with exogenous 5-HT and was not reduced by reserpine and 6-OHDA. When no exogenous 5-HT was added, the atria 5-HT levels measured by HPLC were unchanged in all our experimental groups. These results were close to those we found in atria immediately after the rats sacrifice in the absence of any treatment. This 5-HT may be located into residual platelets that the preparations can contain. In conclusion, 5-HT is taken into the storage vesicles of the rat atrial noradrenergic nerves. The way used to cross the nerve membranes does not implicate the classic NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation of 5-HT of an unknown mechanism was also noticed. A part of this extraneuronal accumulation is intracellular. REFERENCES
Allen G. S., Henderson L. M., Chou S. N. and French L. A. (1974) Cerebral arterial spasm. Part 2: in vitro contractile activity of serotonin in human serum and CSF on the canine basilar artery, and its blockade by methysergide and phenoxybenzamine. J. Neurosurg. 40, 442-450. Angus J. A., Brazenor R. M. and Le Duc M. A. (1982) Verapamih a selective antagonist of constrictor substances in coronary artery: implication for variant angina. Clin. exp. Pharmac. Physiol. 6, 15-28. Bove A. A. and Dewey J. D. (1983) Effects of serotonin and histamine on proximal and distal coronary vasculature in dogs: comparison with alpha-adrenergic stimulation. Am. J. Cardiol. 52, 1333-1339. Bryan L. J., O'Donnell S. R. and Williams A. M. (1989) Dissipation mechanisms for 5-hydroxytryptamine in the coronary circulation of the isolated perfused heart of the rat. Br. J. Pharmac. 97, 329-338. Buchan P., Lewis A. J. and Sugrue M. F. (1974) A comparison of the accumulation of noradrenaline and 5-hydroxytryptamine into arterial smooth muscle. Br. J. Pharmac. 52, 132P-133P. Claasen V. (1983) Review of the animal pharmacology and pharmacokinetics of fluvoxamine. Br. J. clin. Pharmac. 15, 349S-355S. Fukuda S., Su C. and Lee T. J.-F. (1986) Mechanisms of extraneuronal serotonin uptake in the rat aorta. J. Pharmac. exp. Ther. 239, 264-269. Halpern A., Kuhn P. H., Shaftel H. E., Samuels S. S. and Shaftel N. (1960) Raynaud's disease, Raynaud's phenomenon, and serotonin. Angiology 11, 151-167. Iversen L. L. and Salt P. J. (1970) Inhibition of catecholamine uptake2 by steroids in the isolated rat heart. Br. J. Pharmac. 40, 528-530.
Uptake of 5-HT in rat isolated atria Iwasawa Y. and Gillis C. N. (1974) Pharmacological analysis of norepinephrine and 5-hydroxytryptamine removal from the pulmonary circulation: differentiation of uptake sites of each amine. J. Pharmac. exp. Ther. 188, 386-393. Junod A. F. (1972) Uptake, metabolism and efltux of 14C-5-hydroxytryptamine in isolated perfused rat lungs. J. Pharmac exp. Ther. 183, 341-355. Kawasaki H. and Takasaki K. (1984) Vasoconstrictor response induced by 5-hydroxytryptamin¢ released from vascular adrenergic nerves by periarterial nerve stimulation. J. Pharmac. exp. Ther. 229, 816-822. Le Fur G. and Uzan A. (1977) Effects of 4-(3-indolyl-alkyl) piperidine derivatives on uptake and release of noradrenaline, dopamine and 5-hydroxytryptamine in rat brain synaptosomes, rat heart and human blood platelets. Biochem. Pharmac. 26, 497-503. Nedergaard O. A. (1988) Catecholamines: regulation, re!ease and inactivation. Pharmac. Toxic. 63 (Suppl. I), 5-8. Paiva M. Q., Caramona M. and Osswald W. (1984) Intraand extraneuronal metabolism of 5-hydroxytryptamine in the isolated saphenous vein of the dog. NaunynSchmiedeberg's Arch. Pharmac. 325, 62-68. Raskin N. H. (1981) Pharmacology of migraine. A. Rev. Pharmac. Toxic. 21, 463-478.
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Shepro D., Batbouta J. C., Robblee L. S., Carson M. P. and Belamarich F. A. (1975) Serotonin transport by cultured bovine aortic endothelium. Circulation Res. 36, 799-806. Stahl S. M. and Meltzer H. Y. (1978) A kinetic and pharmacological analysis of 5-hydroxytryptamine transport by human ptatelets and platelet storage granules: comparison with central serotonergic neurons. J. Pharmac exp. Ther. 205, 118-132. Stranden E., Roald O. K. and Krohg K. (1982) Treatment of Raynaud's phenomenon with the 5-HT2-receptor antagonist ketanserin. Br. med. J. 285, 1069-1071. Theron J. J., Biagio R., Meyer A. C., Boekkooi S. and Seegers J. C. (1978) The effect of a serotonin inhibitor on the serotonin content and ultra-structure of rat atria and ventricles with special reference to atrial granules. Life Sci. 23, 111-120. Thoa N. B., Eccleston D. and Axelrod J. (1969) The accumulation of ~4C-serotonin in the guinea-pig vas deferens. J. Pharmac. exp. Ther. 169, 68-73. Verbeuren T. J., Jordaens F. H. and Herman A. G. (1983) Accumulation and release of [3H]-5-hydroxytryptamine in saphenous veins and cerebral arteries of the dog. J. Pharmac. exp. Ther. 226, 579-588.