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Adenosine contracts the isolated rat tail artery by releasing endogenous-5-hydroxytryptamine
European Journal of Pharmacologv, 76 ( 1981 ) 275-277 Elsevier/North-Holland Biomedical Press
275
Short communication
ADENOSINE CONTRACTS THE ISOLATED RAT TAIL ARTERY BY RELEASING ENDOGENOUS 5-HYDROXYTRYPTAMINE C H R I S T I N E M. B R O W N and M I C H A E L G. COLLIS *
Bioscience I1 Department, Pharmaceuticals Division, I.C.I. Alderlev Park, Macclesfield, Cheshire, U.K. Received 7 October 1981, accepted 13 October 1981
C.M. B R O W N and M.G. COLLIS, Adenosine contracts the isolated rat tail artery by re~easing endogenous 5-hvdroxvtrr'ptamine European J. Pharmacol. 76 (1981) 275-277. Adenosine ( 1 0 - ' * - 1 0 -3 M) contracted the isolated rat tail artery. This effect exhibited rapidly developing tachyphylaxis. Methysergide (10 7 M) prevented the contractile response to adenosine and 5-hydroxytryptamine (5-HT) without affecting that to noradrenaline. Pretreatment of rats with parachlorophenylalanine (PCPA) abolished the contraction to adenosine (5 × 1 0 - 4 M). Responses to 5-HT and noradrenaline were not significantly affected. These results indicate that the contractile response of the rat tail artery induced by adenosine is probably mediated by endogenous 5-HT. Adenosine
5-HT
Rat
Artery
Contraction
I. Introduction
2. Materials and methods
Adenosine causes vasodilation in most mammalian vascular beds (Burnstock, 1980). In addition this purine usually causes relaxation of isolated blood vessels (Vanhoutte, 1978). However, in the perfused femoral vascular bed of the rat adenosine and adenine nucleotides can elicit a prominent vasoconstriction which appears to be mediated by a tryptaminergic mechanism (Sakai and Akima, 1977; 1978; Sakai, 1978; Sakai et al., 1979). The effects of purines on the rat tail artery have not been described, though an interaction between adenosine and hydralazine has recently been reported in this tissue (Worcel et al., 1980). When the effect of adenosine on the rat isolated tail artery was investigated in the present study, a contraction was observed, the nature of which was investigated.
Alderley Park Wistar rats (200-300 g) of either sex were killed by cervical dislocation. The tail arteries were excised and placed in physiological salt solution (PSS) and cut helically into strips. Two strips were prepared from each artery. The strips were mounted vertically in 20 ml tissue baths containing PSS. The upper ends of the strips were connected to Pioden UFI isometric strain gauge transducers, (range -+56 g, Pioden Instruments, Graham Bell House, Roper Road, Canterbury) and the resting tension adjusted to 500 mg. The PSS was maintained at 37°C and aerated with 95% 02 and 5% CO 2. The composition of the solution was as follows (mmol/1): NaC1 118.4; KCI 4.7; KHePO 4 1.2; CaC12 • 2 HzO 2.5; NaHCO 3 25.0; glucose 5.5; MgSO 4 1.2 and CaNa 2EDTA 0.03. Depletion of endogenous 5-HT was carried out by intraperitoneal injection of parachlorophenylalanine (Koe and Weissman, 1966) (300 m g / k g in 0.5% Tween 80). The animals were killed 24 h after the injection. The following drugs were used: adenosine (Sigma), 5-HT creatine sulfate (Sigma), methyser-
* To whom correspondence should be addressed.
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
276
gide bimaleate (Sandoz), (-)-noradrenaline bitartrate (Sigma) and parachlorophenylalanine methyl ester (Sigma). Doses are given as the final molar concentration in the organ bath. All data are expressed as means ± S.E. The results were processed statistically using Student's t-test. Differences were accepted as significant at the 0.05 level of probability.
(a)
aooog [
. m
m
1 rain adenosine 1 x 10"4M
(b)
3 x lO'4M
3x
10"4M
Control
1 min adenosine 5x
3. Results
noredrenaline
IO-4M
5x IO-7M
5-HT 5 x 10"6M
Mathysergide
A single application of adenosine (10 - 4 10-3 M) to the rat tail artery produced a contractile response (range: 30-240 mg) while upon consecutive applications the contractions diminished progressively and by the third application had disappeared (fig. l a). Since tachyphylaxis developed to these concentrations of adenosine only one application of the agonist was used in each of the following experiments. The marked tachyphylaxis to the contractile response of adenosine suggested that it might be due to an endogenous substance released from its peripheral store. In this respect similar tachyphylactic responses to adenosine have been recorded in the femoral vasculature of the rat, and were attributed to 5-HT (Sakai et al., 1979). Thus the effect of methysergide on the contractile response to adenosine was examined. Incubation with methysergide (10 -7 M) for 30 min markedly reduced the contractile response to adenosine (5 × 10-4 M) (control: 66 ± 16 mg, methysergide: 4 ± 4 mg, n = 5 : P < 0 . 0 2 ) attenuated that to 5-HT (5 × 10-6 M) (control: 300 ± 30 mg, methysergide: 30 ± 10 mg: P < 0.001) but did not significantly affect that to noradrenaline (5 X 10-TM) (control: 2 3 0 ± 5 0 mg, methysergide: 190 ± 70 mg) (fig. lb). In rats pretreated with PCPA, which specifically depletes 5-HT stores (Koe and Weissman, 1966), the contraction to adenosine (5 × 10 -4 M) was markedly reduced (7 ± 5 mg, n = 10: P < 0.01) (fig. lc). Responses to 5-HT (5 X 10 -6 M) and noradrenaline (NA) (5 X 10-7 M) application were not significantly different from those in control animals (NA control: 230 ± 50 mg, n -- 5; PCPA: 247 ± 43 mg, n = 10; 5-HT control: 300 ± 30 mg, n = 5; PCPA: 207 ± 40 mg, n = 10). Consecutive applications of 5-HT (5
,°°,E_____ I rain adenosine 5 x I0-~ (¢)
J noradrenaline 5 x lO'7M
5-HT 5 x lO'6M
PCPATreated
1 min (d)
r-~
adenosine
5- HT
noradrenaline
5 x lO'4M
5 x lO'6M
5 x lO'TM
.
.
.
.
lmin 5-HT 5 x lO'6M
5 x IO-6M
5 x lO'6M
5 x IO'6M
r'ig. I. Response of the rat tail artery to adenosine, noradrenaline and 5-HT. (a) Consecutive applications of adenosine to a rat tail artery strip. Horizontal bars represent the period of exposure to the drug. (b) Responses of a paired set of tail artery strips to adenosine, noradrenaline and 5-HT. Top trace, control preparation; lower trace, preparation incubated with methysergide (10 - 7 M) for 30 min. (c) Responses of a tail artery strip from a rat treated 24 h previously with PCPA (300 m g / k g i.p.), to adenosine, 5-HT and noradrenaline. (d) Consecutive applications of 5-HT to a rat tail artery preparation.
X 10 - 6 M) did not exhibit the m a r k e d tachyphylaxis observed with adenosine (fig. 1d).
4. Discussion
These results indicate that the contraction of the rat isolated tail artery evoked by adenosine is
277
probably mediated by endogenous 5-HT. This is borne out by two lines of evidence. Firstly, the contractile response to adenosine was attenuated by methysergide, in a concentration that antagonised the effects of 5-HT, without significantly affecting those to NA. Secondly, after pretreatment with PCPA, the contractile response to adenosine was markedly reduced while responses to N A and 5-HT were unaffected. In the rat isolated perfused hind-limb a single injection of adenosine has also been reported to evoke a vasoconstriction that is diminished by pretreatment with reserpine or methysergide (Sakai and Akima, 1978). Sakai et al. (1979) have further demonstrated that the efflux of 5-HT from this preparation is increased by adenosine, indicating that the autocoid is responsible for the vasoconstriction. The present results, and those of Sakai and Akima (1978) demosntrate that the vasoconstrictor response evoked by adenosine rapidly develops tachyphylaxis. Thus it would appear that one or two applications of the purine are sufficient to release all the available 5-HT from its peripheral store. The nature of the peripheral store remains to be investigated. One possibility is that adenosine causes the degranulation of mast cells which contain significant amounts of 5-HT in the rat (Erspamer, 1966).
Acknowledgement The authors wish to thank Mr. T.P. Blackburn for his technical assistance.
References Burnstock, G., 1980, Cholinergic and purinergic regulation of blood vessels, in: Handbook of Physiology, Sec. 2. The Cardiovascular System, Vol. II. Vascular Smooth Muscle, eds. D.F. Bohr, A.D. Somlyo, H.W. Sparks and S.R. Geiger (Am. Physiol. Soc., Waverley Press, Baltimore) p. 567. Erspamer, V., 1966, Occurrence of indolealkylamines in nature, in: 5-Hydroxytryptamine and Related Indolealkylamines, Handbook of Experimental Pharmacology XIX, ed. V. Erspamer (Springer-Verlag, New York) p. 132. Koe, B.K. and A. Weissman, 1966, p-Chlorophenylalanine. A specific depletor of brain serotonin, J. Pharmacol. Exp. Ther. 154, 499. Sakai, K., 1978, Tryptaminergic mechanism participating in induction of vasoconstriction by adenine nucleotides, adenosine, IMP and inosine in the isolated and bloodperfused hindlimb preparation of the rat, Jap. J. Pharmacol. 28, 579. Sakai, K. and M. Akima, 1977, Tryptamine vasoconstriction induced by adenosine in the femoral vascular bed of rat, Jap. J. Pharmacol. 27, 908. Sakai, K. and M. Akima, 1978, Vasoconstriction after adenosine and inosine in the rat isolated hindlimb abolished by blockade of tryptaminergic mechanisms, NaunynSchmiedeb. Arch. Pharmacol. 302, 55. Sakai, K., M. Akima and H. Matsushita, 1979, Femoral vascular responses to purine and pyrimidine derivatives: release of 5-hydroxytryptamine by purine derivatives in isolated, cross-circulated rat hindlimb, Jap. J. Pharmacol. 29, 243. Vanhoutte, P.M., 1978, Heterogeneity in vascular smooth muscle, in: Microcirculation, Vol. II, eds. G. Kaley and B.M. Altura (University Park Press, Baltimore). p. 181. Worcel, M., B. Saiag and C. Chevillard, 1980, An unexpected mode of action for hydralazine (HYD), T.I.P.S. I, 136.
275
Short communication
ADENOSINE CONTRACTS THE ISOLATED RAT TAIL ARTERY BY RELEASING ENDOGENOUS 5-HYDROXYTRYPTAMINE C H R I S T I N E M. B R O W N and M I C H A E L G. COLLIS *
Bioscience I1 Department, Pharmaceuticals Division, I.C.I. Alderlev Park, Macclesfield, Cheshire, U.K. Received 7 October 1981, accepted 13 October 1981
C.M. B R O W N and M.G. COLLIS, Adenosine contracts the isolated rat tail artery by re~easing endogenous 5-hvdroxvtrr'ptamine European J. Pharmacol. 76 (1981) 275-277. Adenosine ( 1 0 - ' * - 1 0 -3 M) contracted the isolated rat tail artery. This effect exhibited rapidly developing tachyphylaxis. Methysergide (10 7 M) prevented the contractile response to adenosine and 5-hydroxytryptamine (5-HT) without affecting that to noradrenaline. Pretreatment of rats with parachlorophenylalanine (PCPA) abolished the contraction to adenosine (5 × 1 0 - 4 M). Responses to 5-HT and noradrenaline were not significantly affected. These results indicate that the contractile response of the rat tail artery induced by adenosine is probably mediated by endogenous 5-HT. Adenosine
5-HT
Rat
Artery
Contraction
I. Introduction
2. Materials and methods
Adenosine causes vasodilation in most mammalian vascular beds (Burnstock, 1980). In addition this purine usually causes relaxation of isolated blood vessels (Vanhoutte, 1978). However, in the perfused femoral vascular bed of the rat adenosine and adenine nucleotides can elicit a prominent vasoconstriction which appears to be mediated by a tryptaminergic mechanism (Sakai and Akima, 1977; 1978; Sakai, 1978; Sakai et al., 1979). The effects of purines on the rat tail artery have not been described, though an interaction between adenosine and hydralazine has recently been reported in this tissue (Worcel et al., 1980). When the effect of adenosine on the rat isolated tail artery was investigated in the present study, a contraction was observed, the nature of which was investigated.
Alderley Park Wistar rats (200-300 g) of either sex were killed by cervical dislocation. The tail arteries were excised and placed in physiological salt solution (PSS) and cut helically into strips. Two strips were prepared from each artery. The strips were mounted vertically in 20 ml tissue baths containing PSS. The upper ends of the strips were connected to Pioden UFI isometric strain gauge transducers, (range -+56 g, Pioden Instruments, Graham Bell House, Roper Road, Canterbury) and the resting tension adjusted to 500 mg. The PSS was maintained at 37°C and aerated with 95% 02 and 5% CO 2. The composition of the solution was as follows (mmol/1): NaC1 118.4; KCI 4.7; KHePO 4 1.2; CaC12 • 2 HzO 2.5; NaHCO 3 25.0; glucose 5.5; MgSO 4 1.2 and CaNa 2EDTA 0.03. Depletion of endogenous 5-HT was carried out by intraperitoneal injection of parachlorophenylalanine (Koe and Weissman, 1966) (300 m g / k g in 0.5% Tween 80). The animals were killed 24 h after the injection. The following drugs were used: adenosine (Sigma), 5-HT creatine sulfate (Sigma), methyser-
* To whom correspondence should be addressed.
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
276
gide bimaleate (Sandoz), (-)-noradrenaline bitartrate (Sigma) and parachlorophenylalanine methyl ester (Sigma). Doses are given as the final molar concentration in the organ bath. All data are expressed as means ± S.E. The results were processed statistically using Student's t-test. Differences were accepted as significant at the 0.05 level of probability.
(a)
aooog [
. m
m
1 rain adenosine 1 x 10"4M
(b)
3 x lO'4M
3x
10"4M
Control
1 min adenosine 5x
3. Results
noredrenaline
IO-4M
5x IO-7M
5-HT 5 x 10"6M
Mathysergide
A single application of adenosine (10 - 4 10-3 M) to the rat tail artery produced a contractile response (range: 30-240 mg) while upon consecutive applications the contractions diminished progressively and by the third application had disappeared (fig. l a). Since tachyphylaxis developed to these concentrations of adenosine only one application of the agonist was used in each of the following experiments. The marked tachyphylaxis to the contractile response of adenosine suggested that it might be due to an endogenous substance released from its peripheral store. In this respect similar tachyphylactic responses to adenosine have been recorded in the femoral vasculature of the rat, and were attributed to 5-HT (Sakai et al., 1979). Thus the effect of methysergide on the contractile response to adenosine was examined. Incubation with methysergide (10 -7 M) for 30 min markedly reduced the contractile response to adenosine (5 × 10-4 M) (control: 66 ± 16 mg, methysergide: 4 ± 4 mg, n = 5 : P < 0 . 0 2 ) attenuated that to 5-HT (5 × 10-6 M) (control: 300 ± 30 mg, methysergide: 30 ± 10 mg: P < 0.001) but did not significantly affect that to noradrenaline (5 X 10-TM) (control: 2 3 0 ± 5 0 mg, methysergide: 190 ± 70 mg) (fig. lb). In rats pretreated with PCPA, which specifically depletes 5-HT stores (Koe and Weissman, 1966), the contraction to adenosine (5 × 10 -4 M) was markedly reduced (7 ± 5 mg, n = 10: P < 0.01) (fig. lc). Responses to 5-HT (5 X 10 -6 M) and noradrenaline (NA) (5 X 10-7 M) application were not significantly different from those in control animals (NA control: 230 ± 50 mg, n -- 5; PCPA: 247 ± 43 mg, n = 10; 5-HT control: 300 ± 30 mg, n = 5; PCPA: 207 ± 40 mg, n = 10). Consecutive applications of 5-HT (5
,°°,E_____ I rain adenosine 5 x I0-~ (¢)
J noradrenaline 5 x lO'7M
5-HT 5 x lO'6M
PCPATreated
1 min (d)
r-~
adenosine
5- HT
noradrenaline
5 x lO'4M
5 x lO'6M
5 x lO'TM
.
.
.
.
lmin 5-HT 5 x lO'6M
5 x IO-6M
5 x lO'6M
5 x IO'6M
r'ig. I. Response of the rat tail artery to adenosine, noradrenaline and 5-HT. (a) Consecutive applications of adenosine to a rat tail artery strip. Horizontal bars represent the period of exposure to the drug. (b) Responses of a paired set of tail artery strips to adenosine, noradrenaline and 5-HT. Top trace, control preparation; lower trace, preparation incubated with methysergide (10 - 7 M) for 30 min. (c) Responses of a tail artery strip from a rat treated 24 h previously with PCPA (300 m g / k g i.p.), to adenosine, 5-HT and noradrenaline. (d) Consecutive applications of 5-HT to a rat tail artery preparation.
X 10 - 6 M) did not exhibit the m a r k e d tachyphylaxis observed with adenosine (fig. 1d).
4. Discussion
These results indicate that the contraction of the rat isolated tail artery evoked by adenosine is
277
probably mediated by endogenous 5-HT. This is borne out by two lines of evidence. Firstly, the contractile response to adenosine was attenuated by methysergide, in a concentration that antagonised the effects of 5-HT, without significantly affecting those to NA. Secondly, after pretreatment with PCPA, the contractile response to adenosine was markedly reduced while responses to N A and 5-HT were unaffected. In the rat isolated perfused hind-limb a single injection of adenosine has also been reported to evoke a vasoconstriction that is diminished by pretreatment with reserpine or methysergide (Sakai and Akima, 1978). Sakai et al. (1979) have further demonstrated that the efflux of 5-HT from this preparation is increased by adenosine, indicating that the autocoid is responsible for the vasoconstriction. The present results, and those of Sakai and Akima (1978) demosntrate that the vasoconstrictor response evoked by adenosine rapidly develops tachyphylaxis. Thus it would appear that one or two applications of the purine are sufficient to release all the available 5-HT from its peripheral store. The nature of the peripheral store remains to be investigated. One possibility is that adenosine causes the degranulation of mast cells which contain significant amounts of 5-HT in the rat (Erspamer, 1966).
Acknowledgement The authors wish to thank Mr. T.P. Blackburn for his technical assistance.
References Burnstock, G., 1980, Cholinergic and purinergic regulation of blood vessels, in: Handbook of Physiology, Sec. 2. The Cardiovascular System, Vol. II. Vascular Smooth Muscle, eds. D.F. Bohr, A.D. Somlyo, H.W. Sparks and S.R. Geiger (Am. Physiol. Soc., Waverley Press, Baltimore) p. 567. Erspamer, V., 1966, Occurrence of indolealkylamines in nature, in: 5-Hydroxytryptamine and Related Indolealkylamines, Handbook of Experimental Pharmacology XIX, ed. V. Erspamer (Springer-Verlag, New York) p. 132. Koe, B.K. and A. Weissman, 1966, p-Chlorophenylalanine. A specific depletor of brain serotonin, J. Pharmacol. Exp. Ther. 154, 499. Sakai, K., 1978, Tryptaminergic mechanism participating in induction of vasoconstriction by adenine nucleotides, adenosine, IMP and inosine in the isolated and bloodperfused hindlimb preparation of the rat, Jap. J. Pharmacol. 28, 579. Sakai, K. and M. Akima, 1977, Tryptamine vasoconstriction induced by adenosine in the femoral vascular bed of rat, Jap. J. Pharmacol. 27, 908. Sakai, K. and M. Akima, 1978, Vasoconstriction after adenosine and inosine in the rat isolated hindlimb abolished by blockade of tryptaminergic mechanisms, NaunynSchmiedeb. Arch. Pharmacol. 302, 55. Sakai, K., M. Akima and H. Matsushita, 1979, Femoral vascular responses to purine and pyrimidine derivatives: release of 5-hydroxytryptamine by purine derivatives in isolated, cross-circulated rat hindlimb, Jap. J. Pharmacol. 29, 243. Vanhoutte, P.M., 1978, Heterogeneity in vascular smooth muscle, in: Microcirculation, Vol. II, eds. G. Kaley and B.M. Altura (University Park Press, Baltimore). p. 181. Worcel, M., B. Saiag and C. Chevillard, 1980, An unexpected mode of action for hydralazine (HYD), T.I.P.S. I, 136.