- Email: [email protected]
Vasoconstrictor effects of serotonin in the isolated superior mesenteric artery of cat
Gen. Pharmac. Vol. 12. pp. 97 to 101
.
0306-3623/81/030097-05502.00/0
© Pergamon Press Ltd i981. Printed in Great Britain
VASOCONSTRICTOR EFFECTS OF SEROTONIN IN THE ISOLATED SUPERIOR MESENTERIC ARTERY OF CAT J. MARfN, M. ARIAS,M. SALMCES,C. F. SANOtEZ and U RECIO Departamento de Farmacologia y Terap6utica, Facultad de Medicina, Universidad Aut6noma de Madrid, C/Arzobispo Morcillo 4. Madrid-34. Spain (Received 28 July 1980) Abstraet--l. 5-Hydroxytryptamine (5-HT) evoked dose-dependent contractions in the isolated superior mesenteric artery of cat. 2. LSD and LSD plus phentolamine significantly and markedly reduced the vasoconstriction induced by 5-HT, respectively. However, phentolamine and reserpine pretreatment did not. 3. 5-HT (10-s-10 -3 M), K + and tyramine produced tritium release from vessels prelabelled with l-3H]noradrenaline. Ca2+-deprivation did not modify the release caused by 5-HT and tyramine, although the tritium secretion evoked by K + was abolished in a Ca 2+-free medium. 4. These results indicate that the vasoconstriction induced by 5-HT is mainly mediated through 5-HT receptors. The noradrenaline release by 5-HT is different to that of K + (Ca2+-dependent depolarization) and similar to that of tyramine (Ca2+-independent process).
dent) the influence of extraceilular Ca 2+ on the release caused by 5-HT, tyramine or K + was analyzed.
INTRODUCTION
It is krlown that serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor agent that circulates in association with the blood platelets and is released from them during their aggregation producing local vasoconstriction. The fact that the presence of 5-HT in several vessels has been demonstrated (Berkowitz et al., 1975; Jarrot et al., 1975; Reinhard et al., 1979) suggests the possibility that this amine might be involved in the maintenance of vascular tone. Thus, recently McGrath (1977) has observed that low doses of 5-HT (10 - s M) decrease the noradrenaline release evoked by transmural electric-stimulation of canine saphenous vein and tibial arteries. On the other hand, it has been demonstrated that the vasoconstrictor effects of 5-HT in several vessels are mediated by 5-HT receptors (McGrath, 1977; Edvinsson et al., 1978; Humphrey, 1978; Marin et al., 1979a). Besides this direct effect, this amine may also release noradrenaline from several tissues (Fillion et al., 1971; Fozard & Mwaluko, 1976; McGrath, 1977; Starke & Weitzell, 1978; Marin et al., 1979b), contributing this indirect component to the overall actions of 5-HT. Furthermore, it has been shown that this release is Ca2+-dependent on isolated rabbit heart and cat pial vessels (Fozard & Mwaluko, 1976; Marin & S~inchez, 1980) while that in rabbit pulmonary artery seems to be tyramine-like (Starke & Weitzell, 1978). The purpose of the present work was to study in cat superior mesenteric artery the participation, in the contraction evoked by 5-HT, of two effects of this amine: a direct one on 5-HT receptors and an indirect adrenergic one, which involves noradrenaline liberation from adrenergic stores. In order to observe whether this liberation is tyramine-like (i.e. Ca 2+independent) or potassium (K +)-like (i.e. Ca 2+-depen-
MATERIALS
AND METHODS
Cats (1.5-3kg) were anesthetized with 35mg/kg of sodium pentobarbital administered intraperitoneaily and killed by bleeding. The superior mesenteric artery was removed and dissected out and cut into cylindrical segments 4 mm in length. The cylinder was set up in an organ bath kept at 37°C containing 6 ml of Krebs-Henseleit solution (KHS) continuously bubbled with 95% 02 and 5% CO2 to give a pH of 7.3-7.4. Two stainless steel pins were introduced through the lumen of the arterial segment. One pin was fixed to the organ bath wall while the other one was connected to a strain gauge for isometric tension recording. The latter pin is in parallel position with the former and movable, thus permitting the application of resting tension in a perpendicular plane to the long axis of the vascular cylinder. The recording system included a force-displacement transducer (Grass FT03C) and a Grass Model 7D Poligraph. The composition of KHS was (raM): NaCI, 115; KCI, 4.6; CaCi2, 2.5; KH2PO4, 1.2; MgSO,.7 H20, 1.2; NaHCO3, 25; glucose, 11.1; disodium salt of ethylenediamine tetraacetic acid (Na2EDTA), 0.03. A resting tension of lg was applied to the tissue and reajusted every 15min during 90--120min equilibration period before cumulative dose-response curves for 5-HT were made. Drugs were disolved in physiological saline solution containing 0.01% (w/v) ascorbic acid. When phentolamine and lysergic acid diethylamide (LSD), a adrenergic and tryptaminergic blockers, respectively, were used, they were added to the bath 10rain before 5-HT and allowed to remain in contact with the tissue throughout the determination of dose-response relationship. Control and experimental responses were obtained from separate vascular preparations. In 7 cats 5 mg of reserpine was intraperitoneally administered one day before the experiment. 97
98
J. MARfN et al.
To measure the endogenous noradrenaline content of the arteries, the vessels were blotted, weighed and homogenized in 1 ml of 0.1 perchloric acid. The homogenates were centrifuged at 15,0000 for 15min and the noradrenaline content of supernatant estimated according to the sensitive radioenzymatic assay of Henry et al. (1975). The results were expressed as #g/g of wet tissue. Tritium efflux The arteries were placed in a Petri dish, which contained ice-cold KHS and the blood contained in them was removed. Afterwards, the vessels were placed on a cylindrical nylon net and put into a beaker containing 4 ml of oxygenated KHS. After a 15 rain equilibration period at 37°C, the tissues were then exposed to (±) EaH]noradrenaline ([3H]NA, 2 x 10 -7 M, sp. act. 10.7Ci/mmol) for 30rain and thoroughly washed with fresh KHS at 10 rain intervals during a l l 2 m i n period. To estimate the spontaneous tritium release, the arteries were successively immersed in two vials containing 2 ml of fresh KHS for 3 rain periods. The drug.evoked release was analyzed by transferring the tissue to another four vials each one containing 2 ml of KHS with the appropiate concentration of the drugs studied (75 mM K +, 10-5-10 -3 M 5-HT); finally the arteries were again exposed to fresh KHS in another five vials in order tO recover the basal level of tritium efflux. Total radioactivity present in the media was analyzed by adding 0.5 ml of each sample to 10 ml of Bray's solution (Bray, 1960) and it was measured in a Nuclear Chicago liquid scintillation counter, model Isocap 300. The pre-drug effiux (at 115 rain) was given the value 1 and the release caused by 5-HT and K + was calculated with respect to this. The tritium-efflux for this value 1 was expressed as cpm/mg of wet tissue. In some experiments 80 rain after the incubation period, the arteries were washed with KHS without Ca 2+ to the end of the experiment. The. . Jfollowing drugs were used: 5-hydroxytryptamine . . . . creatmme sulfate (Sigma), tyramme hydrochlonde (Sigma), potassium chloride (Merck), (_+)[3H]noradrenaline hydrochloride (Radiochemical Centre, Amersham), phentolamine methanesulfonate (Ciba), reserpine (Ciba), lysergic acid diethylamide (Sandoz). Statistical analysis was done according to conventional procedures (Snedecor & Cochran, 1967). RESULTS
A • Control ( 8 ) / ~
Bcontrol[8) I l !
• L S O re)
• Phento+(8)
ZOOO
E=O '--
1000
S.S i
10" 1
10-7
10-0
tO-S
10-0
10-1
10-~
10-S
5-HT(x2.4M ) Fig. 1. Effect of 10 -s M LSD (A) and 10-6M phentolamine (B) on the dose-response curve to 5-HT in superior mesenteric artery of cat. Figures in parentheses indicate the number of arterial segments used. Each point represents the mean _+ SEM.
arteries showed a rapid initial fall which then became slower and practically was levelled off after 100 min of washout. 5-HT (I0 -5, 10 -4 and 10 -3 M) produced a dose-dependent increase in the radioactivity release over the basal level. The peak of this release was reached at 9 min of incubation with 10 - s or 10 -4 M 5*HT and at 12rain for 1 0 - a M 5-HT (Fig. 3A). This
last concentration caused an important amount of tritium secretion and was choosen in the study of Ca 2 +dependence. Ca 2 + :deprivation did not modify the secretory effects of 5-HT (Fig. 3B). A similar pattern of tritium release was obtained with tyramine (10 -4 or 10 -3 M) in normal KHS, and in the OCa2+-KHS this tritium release was also not altered (Fig. 4). On the other hand, 75 mM K + (in which N a + was changed by K + to maintain the osmolarity) induced a great increase in the radioactivity liberation. The peak of secretion was obtained in the first 3 rain of incubation period (Fig. 5). In contrast to what occurred with 5-HT, in a Ca 2 +-free medium this release was practically abolished (P < 0.01) (Fig. 5).
Dose.response curves
Serotonin evoked dose-dependent increases in tension in mesenteric artery of cat. Tachyphylaxis was not developed after repeated trials of 5-HT, though there was an increase, but not significant, in the response to various doses of this amine. The vasoconstriction induced by 5-HT was significantly reduced in pre~nce of 10 - s M LSD, at all doses used (P < 0.05) (Fig. 1A). Although 1 0 - 6 M phentolamine and reserpine pretreatement diminished the contraction evoked by 5-HT this reduction was not significant with respect to control arteries (Figs 1B, 2B). The simultaneous use of phentolamine and LSD practically abolished the response induced by 5-HT (P < 0.001) (l~ig, 2A). The noradrenaline content in control arteries (1.68 +_ 0.11/~g/g, four determinations) was significantly decreased by reserpine pretreatment (0.02 + 0.02 #g/g, three determinations) (P < 0.02). Tritium eJflux The spontaneous tritium release from mesenteric
A *Control(8) [ ! T QLSD+Phento,/ t !
B
.Control(8) [ r , oReserp i n e ~ 7 ~
ZOO0
P =0
1000
O
E
i
10" l
IQ-?
104
10 "6
10-|
10"7
10-0
10-0
5-HT( x 2.4M) Fig. 2. Effect of 10 -s M plus 10 -6 M phentolamine (A) and pretreatment with reserpine (B) on the dose-response curve to 5-HT in superior mesenteric artery of cat. Figures in parentheses indicate the number of arterial segments used. Each point represents the mean + SEM.
Serotonin and mesenteric arteries A • 10"3M (4) o 10~M(3)"
99
B • KHS (4) o OCa
T
gl
i x
!
..,e
5-HT
o
5-HTll0-)M)
tm
It/
Pl
Ill
i I~t?
142 r n i N
Fig. 3. Tritium efflux induced by 5-HT from superior mesenteric artery of cat in normal KHS (A) and in Ca2+-frce KHS (B). Tissues were previously labelled with [3H]NA (2 x 10-~ M) and thoroughly washed during a 112 min period before initiation of sample collection. Vials containing 2 ml of bathing solution were collected every 3 min. Each point represents the tritium efflux during a period of 3 rain. The pre-drug effiux (basal level) was taken as 1 and the release caused by 5-HT was calculated with respect to this value. Number of experiments are shown in parentheses. Vertical bars represent SEM. Horizontal lines indicate the time of exposure to 5-HT.
A • 10-=M(6) .^-4 . . . . .
A
B • KHS (5) ,a-=u oOCaZ*_KHS(4) ' u "
T
,
.
== a.
-r- o
~,,,
:
Ty
.z
:
~
i Iz)
.
t,lz
Ji
i ttz
Ty
, j
i |zl
) t4Z m i n
Fig. 4. Tritium efflux evoked by tyramine (Ty) from superior mesenteric artery of cat in normal KHS (A) and Ca2+-free KHS (B). Experimental design as in Fig. 3. Number of experiments are shown in parentheses. Vertical lines represent SEM. Horizontal lines indicate the time of exposure to Ty. The means + SEM of the basal level of tritium release (i.e. pre-drug efflux) in all cases made iri KHS was 275 + 9 cpm/mg. This release was significantly increased ( P < 0.05) in Ca2+-free solution 376 + 18 cpm/mg.
KHS(4)
•
~,
o OCa =*.- K H S ( 3 )
II
t~
/ 2
'1 "r" O
i
11Z
K÷
i
i
12/'
142
rain
Fig. 5. Tritium efflux evoked by K ÷ (75 mM) from superior mesenteric artery of cat in normal KHS and in Ca'*-free KHS. Experimental design as in Fig. 3. Number of experiments are shown in parentheses. Vertical lines represent SEM. Horizontal lines indicate the time of exposure tO K +.
DISCUSSION The present study shows that 5-HT evoked dosedependent contractile responses in mesenteric arteries of cat. This ability to develop tension in response to 5-HT has been observed in different vessels of various animal species (Toda & Fujita, 1973; Appertey et at, 1978; Matin et aL, 1979a). The response induced by 5-HT was decreased by LSD, which indicates that in the vasoconstriction elicited by this amine the 5-HT receptors are involved. Analogous results were obtained by other authors using the same, or others serotonin antagonists (Urquilla et al., 1975; MiillerSchweinitzer, 1976; McGrath, 1977; Edvinsson et al., 1978; Matin et at, 1979a). However, Curro et al. (1978) did not find reduction in the contractile response to 5-HT in canine sapbenous vein with methysergide. It has been reported that serotonin releases noradrenaline from different adrenergicaUy innervated tissues, including blood Vessels (Fillion et al., 1971; Pluchino, 1972; Fozard & Mwaluko, 1976; M a r i n e t al., 1979b; Marin & Sfinchez, 1980). If 5-HT has an indirect sympathetic component in the vasoconstriction caused by 5-HT, it would be expected that reserpine pretreatment, which greatly reduces the noradrenaline content of these arteries, will diminish the
100
J. MARiNet al.
response to 5-HT. However, this was not the case in the present experiments, since the contraction was not significantly reduced in arterial segments from reserpinized animals. These results suggest that 5-HT produces contraction essentially by direct action on 5-HT receptors. The absence of the indirect adrenergic effect in the vasoconstriction evoked by 5-HT agrees with the fact that phentolamine, and ct adrenergic blocker, did not significantly change the response evoked by this amine. However, 5-HT has ability to induce tritium release, though this release was clearly manifested only at high concentrations of this agent, which indicates its unlikely physiological importance. This low noradrenaline release by 5-HT has been shown in cat brain vessels (Marin et al., 1979b) and in peripheral vessels (McGrath, 1977; Strake & WeitzeU, 1977). On the other hand, the tact that LbLJ pros pnento~amine practically abolished the response to 5-HT, suggests that phentolamine potentiates the antagonism by LSD. The explanation of this fact does not seem clear, although it has been reported that either 5-HT can act on 0t adrenoceptors or phentolamine on 5-HT receptors (Apperley et al., 1976), and that both types of receptors have some similarities, probably they are linked by a common active site (Curro et al., 1978). The fact that phentolamine did not significantly block the response induced by 5-HT though it was decreased (Fig. 1B), brings one to think that the above hypothesis could explain the results obtained with the simultaneous use of two blockers. One of the specific requirements for the release of noradrenaline from adrenergic neurons by electrical stimulation or K + is its absolute requirement for extracellular Ca 2+ (Rubin, 1979; Kirpekar, 1975). However, the noradrenaline secretion caused by tyramine is by a different mechanism independent of extracellular Ca 2+ (Thoenen et aL, 1969; Chubb et al., 1972; Thoa et al., 1975; Marin & Shnchez, 1980). The present experiments show that K +, and 5-HT and tyramine release tritium by different mechanisms, since the secretory response caused by K + was Ca 2+dependent and independent of this cation for 5-HT and tyramine. These findings indicate that the serotonin release is tyramine-like, i.e. the amine is taken up by perivascular adrenergic nerves producing displacement of the neurotransmitter. However, K + evoked release is mediated by depolarization of the nerve endings, facilitating Ca 2+-influx and subsequent noradrenaline secretion (Rubin, 1970; Kirpekar, 1975; Thoa et al., 1975). Starke & Weitzell (1978) have obtained a similar conclusion about 5-HT-induced release in rabbit pulmonary artery. Nevertheless, other authors have reported a Ca 2 +-dependent noradrenaline release by serotonin (Fozard & Mwaluko, 1976; Marin & Sfinchez, 1980). SUMMARY
1. 5-Hydroxytryptamine (5-HT) evoked dosedependent contractions in the isolated superior mesenteric artery of cat. Tachyphylaxis was not observed after repeated trials of 5-HT. 2. The vasoconstriction induced by 5-HT was significantly reduced in presence of 10-SM LSD, whereas 10 -6 M phentolamine and pretreatment with
reserpine did not significantly modify it. The simultaneous application of phentolamine and LSD practically abolished the contraction induced by 5-HT. 3. High doses of 5-HT (10-5-10 -3 M), tyramine (10-*or 10-3M) and K + produced tritium release from vessels prelabeUed with I-3H'lnoradrenaline. Ca2+-deprivation did not significantly modify the release caused by 5-HT and tyramine. However, the tritium liberation evoked by potassium chloride (75 raM) was abolished in a Ca 2+-free medium. 4. These data suggest that the vasoconstrictor effects of 5-HT is essentially due to its direct action on 5-HT receptors. In addition, high concentrations of 5-HT release noradrenaline by a mechanism distinct from that found for K + (Ca 2+-dependent depolarization) and similar to that of tyramine (CaZ+-dependent process involving neuronal uptake and stoichiometric displacement). Acknowledgements--This work was supported in part by Ministerio de Sanidad and Comisi6n Asesora de Investigaci6n Cientifica y T6cnica. REFERENCES
APPERLEY E., HUMPHREYP. P. A. & LEVY G. P. (1976) Receptors for 5-hydroxytryptamine and noradrenaline in rabbit isolated ear artery and aorta. Br. J. Pharmac. 58, 211-221. BERKOWITZB. A., LEE C. H. • SPECTORS. (1975) Dispostion of serotonin in the rat blood vessels and heart. Clin. expl. Pharmac. Physiol. 1,397-400. BRAYG. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillator counter. Analyt. Biochem. 1,279-285. CHUBBI. W., DE POTTERW. P. & DE SCHAEPDRYVERA. F. (1972) Tyramine does not release noradrenaline from splenic nerve by exocytosis. Naunyn-Schmiedeberg's Arch. Pharmak. 274, 281-286. CURRO F. A., GREENBERG S., VERBEUREN T. J. d~ VANHOUT~ P. M. (1978) Interaction between alpha adrenergic and serotonergic activation of canine saphenous veins. J. Pharmac. expl. Ther. 207, 936-949. EDVINSSONL., HARDEBOJ. E. ,~" OWMANC. (t978) Pharmacological analysis of 5-hydroxytryptamine receptors in isolated intracranial and extracranial vessels of cat and man. Circulation Res. 42, 143-151. FILLION G. M., LLUCHS. 8/; UVN.~SB. (1971) Release of noradrenaline from the dog heart in situ after intravenous and intracoronary administration of 5-hydroxytryptamine. Acta physiol, scand 83, 115-123. FOZAROJ. R. & MWALUKOG. M. P. (1976) Mechanism of the indirect sympathomimetic effect of 5-hydroxytryptamine on the isolated heart of the rabbit. Br. d. Pharmac. 57, 115-125. HENRYD. P., STARglANB. J., JOHNSOND. G. & WILLIAMS R. H. (1975) A sensitive radioenzymatic assay for norepinephrine in tissues and plasma. Life Sci. 16, 375-384. HUMPHREY P. P. A. (1978) The effects of ~-adrenoceptor antagonists on contractile responses to 5-hydroxytryptamine in dog saphenous vein. Br. d. Pharmac. 63, 671-675. JARROTB., MCQUEENA., GRAFL., LOUISW. J. (1975) Serotonin levels in vascular tissue and the effects of a serotonin synthesis inhibitor on blood pressure in hypertensive rats. Clin. expl. Pharmac. Physiol. Suppl. 2, 201-205. KIRPEKARS. i . (1975) Factors influencing transmission at adrenergic synapses. Prog. Neurobiol. 4, 163-210. LLUCHS., DIEOUEZG., ALBORCHE., ROIZ i . C. & GOMEZ B. (1976) Direct and indirect effects of 5-hydroxytryptamine on cerebral blood vessels. In The Cerebral Ves-
Serotonin and mesenteric arteries sels Wall (EdRed by C~V6S-NAv.~RO J., M^r^io, s F. & Wi)LL~NWEB~. R.), pp. 135-138. Raven Press, New York. M~,tfN J., SXLAfCEsM., MARCOE. J., GOMEZ B. & LLUCH S. (1979a) Analysis of the contractile effect of S-hydroxytryptamine on the isolated posterior communicating artery of the cat. J. Pharm. Pharmac. 31, 456-459. MAgtN J., S^LMcr=s M. & SANeHEzC. F. (1979b) Release of I'SH]noradrenaline induced by 5-hydroxytryptamine from cat pial arteries. J. Pharm. Pharmac. 31, 818-821. M ~ i N J. & SANCHEZC. F. (1980) Release of noradrenaline from cat cerebral arteries by different drugs and potassium. Biochem. Pharmac. 29, 840-842. McGRATH M. A. (1977) 5-hydroxytryptamine and neurotransmitter release in canine blood vessels. Inhibition by low and augmentation by high concentrations. Circulation Res. 41, 428-435. MOLLER-ScHw~INITZERE. (1976) Evidence for stimulation of 5-HT receptors in canine saphenous arteries by ergotamine. Naunyn-Schmiedebery's Arch. Pharmak. 295, 41-44. PLUCmNO S. (1972) Direct and indirect effects of 5-hydroxytryptamine and tyramine on cat smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmak. 272, 189-224. REINARD J. F. JR, LIEBMANJ. E., SCHLOSBERGA. J. & Mos-
101
KOW]TZM. A. (1979) Serotonin neurons project to small blood vessels in the brain. Science 206, 85--87. Ronr~ R. P. (1970) The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. S~D~-COg G. W. & CoomJu'q W. G. (1967) Statistical Methods, 6th edn, Ames. The Iowa State University Press, Iowa. STARKE K. & WEITZELL R. (1978) Is histamine involved in the sympathomimetic effect of nicotine? Naunyn-Schraiedebero's Arch. Pharrnak. 304, 237-248. THOA N. B., WOOTI~IqG. F., AXELRODJ. & KOPIN I. J. (1975) On the mechanism of release of norepinephrine from sympathetic nerves induced by depolarizing agents and sympathomimetic drugs. Molec. Pharmac. 11, 10-18. THOENEN H., HUL~LIMANNA. ,ca HAEFELYW. (1969) Cation dependence of the noradrenaline-releasing action of tyramine. Eur. J. Pharmac. 6, 29-37. TODA N. & FUJITA Y. (1973) Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephrine, and transmural electrical stimulation. Circulation Res. 33, 98-104. URQUILLA P. R., MARCO E. J. & LLUCH S. (1975) Pharmacological receptors of the cerebral arteries of the goat. Blood Vessels 12, 53-67.
.
0306-3623/81/030097-05502.00/0
© Pergamon Press Ltd i981. Printed in Great Britain
VASOCONSTRICTOR EFFECTS OF SEROTONIN IN THE ISOLATED SUPERIOR MESENTERIC ARTERY OF CAT J. MARfN, M. ARIAS,M. SALMCES,C. F. SANOtEZ and U RECIO Departamento de Farmacologia y Terap6utica, Facultad de Medicina, Universidad Aut6noma de Madrid, C/Arzobispo Morcillo 4. Madrid-34. Spain (Received 28 July 1980) Abstraet--l. 5-Hydroxytryptamine (5-HT) evoked dose-dependent contractions in the isolated superior mesenteric artery of cat. 2. LSD and LSD plus phentolamine significantly and markedly reduced the vasoconstriction induced by 5-HT, respectively. However, phentolamine and reserpine pretreatment did not. 3. 5-HT (10-s-10 -3 M), K + and tyramine produced tritium release from vessels prelabelled with l-3H]noradrenaline. Ca2+-deprivation did not modify the release caused by 5-HT and tyramine, although the tritium secretion evoked by K + was abolished in a Ca 2+-free medium. 4. These results indicate that the vasoconstriction induced by 5-HT is mainly mediated through 5-HT receptors. The noradrenaline release by 5-HT is different to that of K + (Ca2+-dependent depolarization) and similar to that of tyramine (Ca2+-independent process).
dent) the influence of extraceilular Ca 2+ on the release caused by 5-HT, tyramine or K + was analyzed.
INTRODUCTION
It is krlown that serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor agent that circulates in association with the blood platelets and is released from them during their aggregation producing local vasoconstriction. The fact that the presence of 5-HT in several vessels has been demonstrated (Berkowitz et al., 1975; Jarrot et al., 1975; Reinhard et al., 1979) suggests the possibility that this amine might be involved in the maintenance of vascular tone. Thus, recently McGrath (1977) has observed that low doses of 5-HT (10 - s M) decrease the noradrenaline release evoked by transmural electric-stimulation of canine saphenous vein and tibial arteries. On the other hand, it has been demonstrated that the vasoconstrictor effects of 5-HT in several vessels are mediated by 5-HT receptors (McGrath, 1977; Edvinsson et al., 1978; Humphrey, 1978; Marin et al., 1979a). Besides this direct effect, this amine may also release noradrenaline from several tissues (Fillion et al., 1971; Fozard & Mwaluko, 1976; McGrath, 1977; Starke & Weitzell, 1978; Marin et al., 1979b), contributing this indirect component to the overall actions of 5-HT. Furthermore, it has been shown that this release is Ca2+-dependent on isolated rabbit heart and cat pial vessels (Fozard & Mwaluko, 1976; Marin & S~inchez, 1980) while that in rabbit pulmonary artery seems to be tyramine-like (Starke & Weitzell, 1978). The purpose of the present work was to study in cat superior mesenteric artery the participation, in the contraction evoked by 5-HT, of two effects of this amine: a direct one on 5-HT receptors and an indirect adrenergic one, which involves noradrenaline liberation from adrenergic stores. In order to observe whether this liberation is tyramine-like (i.e. Ca 2+independent) or potassium (K +)-like (i.e. Ca 2+-depen-
MATERIALS
AND METHODS
Cats (1.5-3kg) were anesthetized with 35mg/kg of sodium pentobarbital administered intraperitoneaily and killed by bleeding. The superior mesenteric artery was removed and dissected out and cut into cylindrical segments 4 mm in length. The cylinder was set up in an organ bath kept at 37°C containing 6 ml of Krebs-Henseleit solution (KHS) continuously bubbled with 95% 02 and 5% CO2 to give a pH of 7.3-7.4. Two stainless steel pins were introduced through the lumen of the arterial segment. One pin was fixed to the organ bath wall while the other one was connected to a strain gauge for isometric tension recording. The latter pin is in parallel position with the former and movable, thus permitting the application of resting tension in a perpendicular plane to the long axis of the vascular cylinder. The recording system included a force-displacement transducer (Grass FT03C) and a Grass Model 7D Poligraph. The composition of KHS was (raM): NaCI, 115; KCI, 4.6; CaCi2, 2.5; KH2PO4, 1.2; MgSO,.7 H20, 1.2; NaHCO3, 25; glucose, 11.1; disodium salt of ethylenediamine tetraacetic acid (Na2EDTA), 0.03. A resting tension of lg was applied to the tissue and reajusted every 15min during 90--120min equilibration period before cumulative dose-response curves for 5-HT were made. Drugs were disolved in physiological saline solution containing 0.01% (w/v) ascorbic acid. When phentolamine and lysergic acid diethylamide (LSD), a adrenergic and tryptaminergic blockers, respectively, were used, they were added to the bath 10rain before 5-HT and allowed to remain in contact with the tissue throughout the determination of dose-response relationship. Control and experimental responses were obtained from separate vascular preparations. In 7 cats 5 mg of reserpine was intraperitoneally administered one day before the experiment. 97
98
J. MARfN et al.
To measure the endogenous noradrenaline content of the arteries, the vessels were blotted, weighed and homogenized in 1 ml of 0.1 perchloric acid. The homogenates were centrifuged at 15,0000 for 15min and the noradrenaline content of supernatant estimated according to the sensitive radioenzymatic assay of Henry et al. (1975). The results were expressed as #g/g of wet tissue. Tritium efflux The arteries were placed in a Petri dish, which contained ice-cold KHS and the blood contained in them was removed. Afterwards, the vessels were placed on a cylindrical nylon net and put into a beaker containing 4 ml of oxygenated KHS. After a 15 rain equilibration period at 37°C, the tissues were then exposed to (±) EaH]noradrenaline ([3H]NA, 2 x 10 -7 M, sp. act. 10.7Ci/mmol) for 30rain and thoroughly washed with fresh KHS at 10 rain intervals during a l l 2 m i n period. To estimate the spontaneous tritium release, the arteries were successively immersed in two vials containing 2 ml of fresh KHS for 3 rain periods. The drug.evoked release was analyzed by transferring the tissue to another four vials each one containing 2 ml of KHS with the appropiate concentration of the drugs studied (75 mM K +, 10-5-10 -3 M 5-HT); finally the arteries were again exposed to fresh KHS in another five vials in order tO recover the basal level of tritium efflux. Total radioactivity present in the media was analyzed by adding 0.5 ml of each sample to 10 ml of Bray's solution (Bray, 1960) and it was measured in a Nuclear Chicago liquid scintillation counter, model Isocap 300. The pre-drug effiux (at 115 rain) was given the value 1 and the release caused by 5-HT and K + was calculated with respect to this. The tritium-efflux for this value 1 was expressed as cpm/mg of wet tissue. In some experiments 80 rain after the incubation period, the arteries were washed with KHS without Ca 2+ to the end of the experiment. The. . Jfollowing drugs were used: 5-hydroxytryptamine . . . . creatmme sulfate (Sigma), tyramme hydrochlonde (Sigma), potassium chloride (Merck), (_+)[3H]noradrenaline hydrochloride (Radiochemical Centre, Amersham), phentolamine methanesulfonate (Ciba), reserpine (Ciba), lysergic acid diethylamide (Sandoz). Statistical analysis was done according to conventional procedures (Snedecor & Cochran, 1967). RESULTS
A • Control ( 8 ) / ~
Bcontrol[8) I l !
• L S O re)
• Phento+(8)
ZOOO
E=O '--
1000
S.S i
10" 1
10-7
10-0
tO-S
10-0
10-1
10-~
10-S
5-HT(x2.4M ) Fig. 1. Effect of 10 -s M LSD (A) and 10-6M phentolamine (B) on the dose-response curve to 5-HT in superior mesenteric artery of cat. Figures in parentheses indicate the number of arterial segments used. Each point represents the mean _+ SEM.
arteries showed a rapid initial fall which then became slower and practically was levelled off after 100 min of washout. 5-HT (I0 -5, 10 -4 and 10 -3 M) produced a dose-dependent increase in the radioactivity release over the basal level. The peak of this release was reached at 9 min of incubation with 10 - s or 10 -4 M 5*HT and at 12rain for 1 0 - a M 5-HT (Fig. 3A). This
last concentration caused an important amount of tritium secretion and was choosen in the study of Ca 2 +dependence. Ca 2 + :deprivation did not modify the secretory effects of 5-HT (Fig. 3B). A similar pattern of tritium release was obtained with tyramine (10 -4 or 10 -3 M) in normal KHS, and in the OCa2+-KHS this tritium release was also not altered (Fig. 4). On the other hand, 75 mM K + (in which N a + was changed by K + to maintain the osmolarity) induced a great increase in the radioactivity liberation. The peak of secretion was obtained in the first 3 rain of incubation period (Fig. 5). In contrast to what occurred with 5-HT, in a Ca 2 +-free medium this release was practically abolished (P < 0.01) (Fig. 5).
Dose.response curves
Serotonin evoked dose-dependent increases in tension in mesenteric artery of cat. Tachyphylaxis was not developed after repeated trials of 5-HT, though there was an increase, but not significant, in the response to various doses of this amine. The vasoconstriction induced by 5-HT was significantly reduced in pre~nce of 10 - s M LSD, at all doses used (P < 0.05) (Fig. 1A). Although 1 0 - 6 M phentolamine and reserpine pretreatement diminished the contraction evoked by 5-HT this reduction was not significant with respect to control arteries (Figs 1B, 2B). The simultaneous use of phentolamine and LSD practically abolished the response induced by 5-HT (P < 0.001) (l~ig, 2A). The noradrenaline content in control arteries (1.68 +_ 0.11/~g/g, four determinations) was significantly decreased by reserpine pretreatment (0.02 + 0.02 #g/g, three determinations) (P < 0.02). Tritium eJflux The spontaneous tritium release from mesenteric
A *Control(8) [ ! T QLSD+Phento,/ t !
B
.Control(8) [ r , oReserp i n e ~ 7 ~
ZOO0
P =0
1000
O
E
i
10" l
IQ-?
104
10 "6
10-|
10"7
10-0
10-0
5-HT( x 2.4M) Fig. 2. Effect of 10 -s M plus 10 -6 M phentolamine (A) and pretreatment with reserpine (B) on the dose-response curve to 5-HT in superior mesenteric artery of cat. Figures in parentheses indicate the number of arterial segments used. Each point represents the mean + SEM.
Serotonin and mesenteric arteries A • 10"3M (4) o 10~M(3)"
99
B • KHS (4) o OCa
T
gl
i x
!
..,e
5-HT
o
5-HTll0-)M)
tm
It/
Pl
Ill
i I~t?
142 r n i N
Fig. 3. Tritium efflux induced by 5-HT from superior mesenteric artery of cat in normal KHS (A) and in Ca2+-frce KHS (B). Tissues were previously labelled with [3H]NA (2 x 10-~ M) and thoroughly washed during a 112 min period before initiation of sample collection. Vials containing 2 ml of bathing solution were collected every 3 min. Each point represents the tritium efflux during a period of 3 rain. The pre-drug effiux (basal level) was taken as 1 and the release caused by 5-HT was calculated with respect to this value. Number of experiments are shown in parentheses. Vertical bars represent SEM. Horizontal lines indicate the time of exposure to 5-HT.
A • 10-=M(6) .^-4 . . . . .
A
B • KHS (5) ,a-=u oOCaZ*_KHS(4) ' u "
T
,
.
== a.
-r- o
~,,,
:
Ty
.z
:
~
i Iz)
.
t,lz
Ji
i ttz
Ty
, j
i |zl
) t4Z m i n
Fig. 4. Tritium efflux evoked by tyramine (Ty) from superior mesenteric artery of cat in normal KHS (A) and Ca2+-free KHS (B). Experimental design as in Fig. 3. Number of experiments are shown in parentheses. Vertical lines represent SEM. Horizontal lines indicate the time of exposure to Ty. The means + SEM of the basal level of tritium release (i.e. pre-drug efflux) in all cases made iri KHS was 275 + 9 cpm/mg. This release was significantly increased ( P < 0.05) in Ca2+-free solution 376 + 18 cpm/mg.
KHS(4)
•
~,
o OCa =*.- K H S ( 3 )
II
t~
/ 2
'1 "r" O
i
11Z
K÷
i
i
12/'
142
rain
Fig. 5. Tritium efflux evoked by K ÷ (75 mM) from superior mesenteric artery of cat in normal KHS and in Ca'*-free KHS. Experimental design as in Fig. 3. Number of experiments are shown in parentheses. Vertical lines represent SEM. Horizontal lines indicate the time of exposure tO K +.
DISCUSSION The present study shows that 5-HT evoked dosedependent contractile responses in mesenteric arteries of cat. This ability to develop tension in response to 5-HT has been observed in different vessels of various animal species (Toda & Fujita, 1973; Appertey et at, 1978; Matin et aL, 1979a). The response induced by 5-HT was decreased by LSD, which indicates that in the vasoconstriction elicited by this amine the 5-HT receptors are involved. Analogous results were obtained by other authors using the same, or others serotonin antagonists (Urquilla et al., 1975; MiillerSchweinitzer, 1976; McGrath, 1977; Edvinsson et al., 1978; Matin et at, 1979a). However, Curro et al. (1978) did not find reduction in the contractile response to 5-HT in canine sapbenous vein with methysergide. It has been reported that serotonin releases noradrenaline from different adrenergicaUy innervated tissues, including blood Vessels (Fillion et al., 1971; Pluchino, 1972; Fozard & Mwaluko, 1976; M a r i n e t al., 1979b; Marin & Sfinchez, 1980). If 5-HT has an indirect sympathetic component in the vasoconstriction caused by 5-HT, it would be expected that reserpine pretreatment, which greatly reduces the noradrenaline content of these arteries, will diminish the
100
J. MARiNet al.
response to 5-HT. However, this was not the case in the present experiments, since the contraction was not significantly reduced in arterial segments from reserpinized animals. These results suggest that 5-HT produces contraction essentially by direct action on 5-HT receptors. The absence of the indirect adrenergic effect in the vasoconstriction evoked by 5-HT agrees with the fact that phentolamine, and ct adrenergic blocker, did not significantly change the response evoked by this amine. However, 5-HT has ability to induce tritium release, though this release was clearly manifested only at high concentrations of this agent, which indicates its unlikely physiological importance. This low noradrenaline release by 5-HT has been shown in cat brain vessels (Marin et al., 1979b) and in peripheral vessels (McGrath, 1977; Strake & WeitzeU, 1977). On the other hand, the tact that LbLJ pros pnento~amine practically abolished the response to 5-HT, suggests that phentolamine potentiates the antagonism by LSD. The explanation of this fact does not seem clear, although it has been reported that either 5-HT can act on 0t adrenoceptors or phentolamine on 5-HT receptors (Apperley et al., 1976), and that both types of receptors have some similarities, probably they are linked by a common active site (Curro et al., 1978). The fact that phentolamine did not significantly block the response induced by 5-HT though it was decreased (Fig. 1B), brings one to think that the above hypothesis could explain the results obtained with the simultaneous use of two blockers. One of the specific requirements for the release of noradrenaline from adrenergic neurons by electrical stimulation or K + is its absolute requirement for extracellular Ca 2+ (Rubin, 1979; Kirpekar, 1975). However, the noradrenaline secretion caused by tyramine is by a different mechanism independent of extracellular Ca 2+ (Thoenen et aL, 1969; Chubb et al., 1972; Thoa et al., 1975; Marin & Shnchez, 1980). The present experiments show that K +, and 5-HT and tyramine release tritium by different mechanisms, since the secretory response caused by K + was Ca 2+dependent and independent of this cation for 5-HT and tyramine. These findings indicate that the serotonin release is tyramine-like, i.e. the amine is taken up by perivascular adrenergic nerves producing displacement of the neurotransmitter. However, K + evoked release is mediated by depolarization of the nerve endings, facilitating Ca 2+-influx and subsequent noradrenaline secretion (Rubin, 1970; Kirpekar, 1975; Thoa et al., 1975). Starke & Weitzell (1978) have obtained a similar conclusion about 5-HT-induced release in rabbit pulmonary artery. Nevertheless, other authors have reported a Ca 2 +-dependent noradrenaline release by serotonin (Fozard & Mwaluko, 1976; Marin & Sfinchez, 1980). SUMMARY
1. 5-Hydroxytryptamine (5-HT) evoked dosedependent contractions in the isolated superior mesenteric artery of cat. Tachyphylaxis was not observed after repeated trials of 5-HT. 2. The vasoconstriction induced by 5-HT was significantly reduced in presence of 10-SM LSD, whereas 10 -6 M phentolamine and pretreatment with
reserpine did not significantly modify it. The simultaneous application of phentolamine and LSD practically abolished the contraction induced by 5-HT. 3. High doses of 5-HT (10-5-10 -3 M), tyramine (10-*or 10-3M) and K + produced tritium release from vessels prelabeUed with I-3H'lnoradrenaline. Ca2+-deprivation did not significantly modify the release caused by 5-HT and tyramine. However, the tritium liberation evoked by potassium chloride (75 raM) was abolished in a Ca 2+-free medium. 4. These data suggest that the vasoconstrictor effects of 5-HT is essentially due to its direct action on 5-HT receptors. In addition, high concentrations of 5-HT release noradrenaline by a mechanism distinct from that found for K + (Ca 2+-dependent depolarization) and similar to that of tyramine (CaZ+-dependent process involving neuronal uptake and stoichiometric displacement). Acknowledgements--This work was supported in part by Ministerio de Sanidad and Comisi6n Asesora de Investigaci6n Cientifica y T6cnica. REFERENCES
APPERLEY E., HUMPHREYP. P. A. & LEVY G. P. (1976) Receptors for 5-hydroxytryptamine and noradrenaline in rabbit isolated ear artery and aorta. Br. J. Pharmac. 58, 211-221. BERKOWITZB. A., LEE C. H. • SPECTORS. (1975) Dispostion of serotonin in the rat blood vessels and heart. Clin. expl. Pharmac. Physiol. 1,397-400. BRAYG. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillator counter. Analyt. Biochem. 1,279-285. CHUBBI. W., DE POTTERW. P. & DE SCHAEPDRYVERA. F. (1972) Tyramine does not release noradrenaline from splenic nerve by exocytosis. Naunyn-Schmiedeberg's Arch. Pharmak. 274, 281-286. CURRO F. A., GREENBERG S., VERBEUREN T. J. d~ VANHOUT~ P. M. (1978) Interaction between alpha adrenergic and serotonergic activation of canine saphenous veins. J. Pharmac. expl. Ther. 207, 936-949. EDVINSSONL., HARDEBOJ. E. ,~" OWMANC. (t978) Pharmacological analysis of 5-hydroxytryptamine receptors in isolated intracranial and extracranial vessels of cat and man. Circulation Res. 42, 143-151. FILLION G. M., LLUCHS. 8/; UVN.~SB. (1971) Release of noradrenaline from the dog heart in situ after intravenous and intracoronary administration of 5-hydroxytryptamine. Acta physiol, scand 83, 115-123. FOZAROJ. R. & MWALUKOG. M. P. (1976) Mechanism of the indirect sympathomimetic effect of 5-hydroxytryptamine on the isolated heart of the rabbit. Br. d. Pharmac. 57, 115-125. HENRYD. P., STARglANB. J., JOHNSOND. G. & WILLIAMS R. H. (1975) A sensitive radioenzymatic assay for norepinephrine in tissues and plasma. Life Sci. 16, 375-384. HUMPHREY P. P. A. (1978) The effects of ~-adrenoceptor antagonists on contractile responses to 5-hydroxytryptamine in dog saphenous vein. Br. d. Pharmac. 63, 671-675. JARROTB., MCQUEENA., GRAFL., LOUISW. J. (1975) Serotonin levels in vascular tissue and the effects of a serotonin synthesis inhibitor on blood pressure in hypertensive rats. Clin. expl. Pharmac. Physiol. Suppl. 2, 201-205. KIRPEKARS. i . (1975) Factors influencing transmission at adrenergic synapses. Prog. Neurobiol. 4, 163-210. LLUCHS., DIEOUEZG., ALBORCHE., ROIZ i . C. & GOMEZ B. (1976) Direct and indirect effects of 5-hydroxytryptamine on cerebral blood vessels. In The Cerebral Ves-
Serotonin and mesenteric arteries sels Wall (EdRed by C~V6S-NAv.~RO J., M^r^io, s F. & Wi)LL~NWEB~. R.), pp. 135-138. Raven Press, New York. M~,tfN J., SXLAfCEsM., MARCOE. J., GOMEZ B. & LLUCH S. (1979a) Analysis of the contractile effect of S-hydroxytryptamine on the isolated posterior communicating artery of the cat. J. Pharm. Pharmac. 31, 456-459. MAgtN J., S^LMcr=s M. & SANeHEzC. F. (1979b) Release of I'SH]noradrenaline induced by 5-hydroxytryptamine from cat pial arteries. J. Pharm. Pharmac. 31, 818-821. M ~ i N J. & SANCHEZC. F. (1980) Release of noradrenaline from cat cerebral arteries by different drugs and potassium. Biochem. Pharmac. 29, 840-842. McGRATH M. A. (1977) 5-hydroxytryptamine and neurotransmitter release in canine blood vessels. Inhibition by low and augmentation by high concentrations. Circulation Res. 41, 428-435. MOLLER-ScHw~INITZERE. (1976) Evidence for stimulation of 5-HT receptors in canine saphenous arteries by ergotamine. Naunyn-Schmiedebery's Arch. Pharmak. 295, 41-44. PLUCmNO S. (1972) Direct and indirect effects of 5-hydroxytryptamine and tyramine on cat smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmak. 272, 189-224. REINARD J. F. JR, LIEBMANJ. E., SCHLOSBERGA. J. & Mos-
101
KOW]TZM. A. (1979) Serotonin neurons project to small blood vessels in the brain. Science 206, 85--87. Ronr~ R. P. (1970) The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. S~D~-COg G. W. & CoomJu'q W. G. (1967) Statistical Methods, 6th edn, Ames. The Iowa State University Press, Iowa. STARKE K. & WEITZELL R. (1978) Is histamine involved in the sympathomimetic effect of nicotine? Naunyn-Schraiedebero's Arch. Pharrnak. 304, 237-248. THOA N. B., WOOTI~IqG. F., AXELRODJ. & KOPIN I. J. (1975) On the mechanism of release of norepinephrine from sympathetic nerves induced by depolarizing agents and sympathomimetic drugs. Molec. Pharmac. 11, 10-18. THOENEN H., HUL~LIMANNA. ,ca HAEFELYW. (1969) Cation dependence of the noradrenaline-releasing action of tyramine. Eur. J. Pharmac. 6, 29-37. TODA N. & FUJITA Y. (1973) Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephrine, and transmural electrical stimulation. Circulation Res. 33, 98-104. URQUILLA P. R., MARCO E. J. & LLUCH S. (1975) Pharmacological receptors of the cerebral arteries of the goat. Blood Vessels 12, 53-67.