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Adenosine 5′-triphosphate, adenosine and endothelium-derived relaxing factor in hypoxic vasodilatation of the heart
European Journal of Pharmacology, 165 (1989) 323-326
323
Elsevier EJP 20402 Short communication
Adenosine 5'-triphosphate, adenosine and endothelium-derived relaxing factor in hypoxic vasodilatation of the heart A l e x a n d r a M. H o p w o o d 1, Jill Lincoln, K a r e n A. K i r k p a t r i c k a n d G e o f f r e y B u r n s t o c k * Department of Anatomy and DevelopmentalBiology and Centrefor Neuroscience, University CollegeLondon, Gower Street, London WCIE 6BT, U.K.
Received 20 April 1989, accepted 25 April 1989
The involvement of ATP in hypoxic vasodilatation was investigated using isolated perfused guinea-pig hearts (Langendorff). Reactive blue 2, a selective P2v-purinoceptor antagonist, attenuated dilatations due to ATP and hypoxia. Hydroquinone, an agent which destroys endothelium-derived relaxing factor, substantially decreased dilatations due to 2-methylthioATP, a potent P2v-purinoceptor agonist, and hypoxia, but not to adenosine. ATP may, therefore, have an important role to play in the initiation of hypoxic dilatation which is mediated by the release of endothelium-derived relaxing factor. Hypoxia; Heart; ATP; Adenosine; Endothelium-derived relaxing factor (EDRF)
1. Introduction Since the early 1960s, adenosine has been regarded as the major agent responsible for the vasodilatation seen during hypoxic perfusion (Berne et al., 1987; Sparks and Gorman, 1987). However, methylxanthines, the antagonists of P1purinoceptors are virtually ineffective at reducing the vasodilatation seen during hypoxia or reactive hyperaemia at concentrations where they fully block the dilatations due to adenosine (Sparks and Gorman, 1987). Further, Ishibashi et al. (1985) showed that the time course of adenosine release from hypoxic heart was too late for the elicited vasodilatation to be due to adenosine. In the present study we have tested the hypothesis that ATP is involved in hypoxic vasodilatation by the use of Reactive blue 2 (RB2), a
1 Present address: Syntex Pharmaceuticals Ltd., Syntex House, St. Ives Road, Maidenhead, Berkshire SL6 1RD, U.K. * To whom all correspondence should be addressed.
compound which has recently been shown to be a relatively specific blocker for vasodilatations, including those in coronary vessels, due to ATP, which are mediated via the P2v-purinoceptor (Hopwood and Burnstock, 1987; Reilly et al., 1987). In addition, we have investigated the idea that endothelium-derived relaxing factor (EDRF) is involved in hypoxic vasodilatation by the use of hydroquinone (a free radical quenching agent).
2. Materials and methods Guinea-pigs (250-500 g) of either sex were injected with heparin (2 500 units i.p.) before being killed by cervical dislocation. The heart was removed and cannulated via the aorta for constant flow perfusion (the average flow was 13.6 + 0.3 m l . min -1, n = 28) according to the method of Langendorff. This has been described in detail previously ( H o p w o o d and Burnstock, 1987). Doses of nucleosides and nucleotides were given in 50 /~1 boluses, delivered over 3 s. The order of
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
324
doses of 2-methylthioATP, adenosine and ATP were varied according to Latin square principles. The dose cycle for all these drugs was 8 min. The heart was made hypoxic by switching from Krebs-Henseleit solution (containing (mM): NaC1 115.3, KC1 4.6, MgSO 4 • 7 H 2 0 1.1, N a H C O 3 22.1, KH2PO 4 1.1, CaC12 2.5, glucose 11.1) equilibrated with 95% 02, 5% CO 2 to one equilibrated with 95% N 2 , 5% C O 2. To minimize mixing, the hypoxic Krebs solution was delivered via a second perfusion system (rate-matched pump) which terminated in syringe needle. As this needle was pushed through a self-sealing rubber injection the main pump was switched off using a remote control device (C.E.E.S. Ltd., Essex). This minimized pressure differences and ischaemia likely to occur as a result of such a switchover. The hypoxic perfusion was continued for 1 rain. When the effect of antagonists was examined, control dose-responses to the purines and a control hypoxic dilatation were first obtained and RB2 or hydroquinone were added to the perfusing solution and allowed to act on the heart for 20 rain. The dose-responses and hypoxic dilatations were then repeated in the presence of the antagonist. The results were calculated as the means _+ S.E.M. and the two-tailed Student's t-test was used to assess significance. At the end of the experiment the heart was removed from the cannula, blotted and weighed. The average heart weights were 1.42 _+ 0.04 g (n = 28). Adenosine, ATP (sodium salt), hydroquinone and RB2 were obtained from Sigma. 2-MethylthioATP was obtained from Research Biochemicals Inc. Heparin was obtained from University College Hospital pharmacy.
3. Results
Occasionally, mainly when high concentrations of 2-methylthioATP were applied, biphasic responses were observed in the guinea-pig heart, as previously reported in the rat (Hopwood and Burnstock, 1987). No comment on relative potencies can be made, as responses to ATP and 2methylthioATP were obtained on different preparations.
30. qE 0) ~20.
5
p s S'~ C3
-log moles ATP Fig. 1. The effect of 1.8/xM RB2 on the vasodilatory responses to A T P and and hypoxia. Control dose-response curve to ATP, solid line; + RB2, broken line. The relative antagonism of this response was 15.8, P < 0.01, n = 9. The control hypoxic dilatation is shown by the blank column, and in the presence of RB2 by the dotted column. Hypoxic dilatation was attenuated by 41%, * P < 0.05, n = 9, two-tailed Student's test.
RB2 at 1.8 # M strongly antagonized the vasodilatory responses to ATP; the relative antagonism, taken as the difference between the values of the pD 2 in the absence and presence of RB2, was 15.8 (fig. 1). At this concentration, RB2 also reduced by over 40% the vasodilatation seen during hypoxic perfusion. Both of these effects of RB2 were statistically significant (P < 0.01 and P < 0.05, respectively). No significant effect of RB2 on left ventricular pressure was observed. The mean perfusion pressure of the preparations (69.6 + 5.4 mmHg, n = 9) was not significantly altered by the administration of RB2 (72.6 + 7.1 mmHg, n = 9). Preliminary experiments revealed that, at a concentration of 50 /xM, hydroquinone decreased left ventricular pressure. The present experiments were therefore carded out with hydroquinone at a concentration of 1 /~M where no direct effect on left ventricular pressure was observed. There was no significant difference in the perfusion pressure of the preparations used in the present study, before (67.3 + 4.3 mmHg, n = 6) or after (68.5 _+ 5.1 mmHg, n = 6) hydroquinone. Hydroquinone
325
IE
20.
g
.~ 10 t-1
c~
-log moles 2Me.S.ATP
Adencer~ 1pMo~
Hymxe
Fig. 2. The effect of 1 g M hydroquinone on the vasodilatory responses of guinea-pig heart vasculature to 2-methylthioATP (2.Me.S.ATP), adenosine and hypoxia. Control responses to 2-methylthioATP, solid line; +hydroquinone, broken line. Control responses to adenosine (left-hand bar chart) and hypoxia (right-hand bar chart), blank colunm; +hydroquinone, dotted column. * P < 0.05, * * P < 0.01, * * * P < 0.001, n = 6, two-tailed Student's t-test.
significantly reduced the 2-methylthioATP-mediated dilations but not those elicited by adenosine (fig. 2). The hypoxic dilation was reduced by well over 50%.
4. Discussion
In the present communication we provide pharmacological evidence in support of the view that ATP is involved in eliciting the vasodilatation seen during hypoxic perfusion. We have previously shown that ATP acts at P2v-purinoceptors in the coronary circulation of the rat, distinct from the Pl-purinoceptors at which adenosine exerts its effects (Hopwood and Burnstock, 1987). RB2, a recently proposed P2y-purinoceptor antagonist, was only one-fifth as effective in attenuating the dilatation caused by adenosine as that caused by ATP in the rat heart (Hopwood and Burnstock, 1987) and had no significant effect on adenosine responses in the rabbit portal vein (Reilly et al., 1987). In the present study, RB2 reduced by over 40% the vasodilatation from hypoxic perfusion, which suggests that ATP is involved in the response seen during hypoxia.
The results are consistent with the view that adenosine can no longer be regarded as the major mediator of hypoxic vasodilatation. It is wellestablished that methylxanthines and adenosine deaminase cannot completely abolish the hypoxic vasodilatation (Sparks and Gorman, 1987) and it has recently been reported that adenosine is released too late for it to be able to account for the response seen during the hypoxic perfusion (Ishibashi et al., 1985). Paddle and Burnstock (1974) have provided evidence that ATP may be an alternative mediator of this response, since they noted that ATP was released in the effluent of guinea-pig hearts when these hearts were made hypoxic, in sufficient amounts to be detectable despite its rapid breakdown by ectoenzymes (only about 1% of ATP introduced into the Langendorff heart survived as ATP; the rest was shown to be largely the breakdown product, adenosine). Busse et al. (1983) have found that endothelial cells are important in mediating hypoxic vasodilatation. In these studies, we used hydroquinone, a compound which, by virtue of its free-radical quenching properties, destroys EDRF (Moncada et al., 1986). Our results reveal that this compound significantly reduced the dilatation due to the selective P2y-purinoceptor agonist, 2-methylthioATP, and to hypoxic perfusion, but not the dilatation due to the Pl-purinoceptor agonist, adenosine. This suggests that EDRF is involved in mediating the vasodilatation due to hypoxia. The involvement of EDRF provides indirect evidence that adenosine cannot possibly be the sole mediator of the dilatation seen when the heart is made hypoxic. The source of the ATP leading to vasodilatation in hypoxia has not been established, however, endothelial cells in culture have been shown to release adenine nucleotides (Pearson and Gordon, 1979). In preliminary experiments, attempts to produce a viable Langendorff heart preparation free of endothelium were unsuccessful. Similarly, the guanylate cyclase blocker, methylene blue, proved to be toxic to the preparation. In the absence of data on the effects of other EDRF inhibitors such as haemoglobin, the possibility of RB2 and hydroquinone having effects on other mechanisms, in addition to those already described here in hypoxia, cannot be totally excluded.
326
In conclusion, the pharmacological evidence presented here suggests that ATP has a role in mediating the response to hypoxia. Hypoxic vasodilatation also appears to depend on the release of EDRF, since hydroquinone also reduced the extent of the hypoxic vasodilatation by over 60%. Since only 60% of hypoxic vasodilatation can be abolished by the use of hydroquinone, this implies that only two-thirds of the response is due to endothelial-mediated mechanism Possibilities for the remaining component include locally released agents such as prostanoids and a contribution from vasodilator nerves. Since 40% of the response is blocked by an ATP antagonist, this suggests that ATP is the predominant agent acting via the endothelium. This leaves the question of what agents are responsible for the other 20%. Possibilities include serotonin, acetylcholine and substance P, which have recently been shown in our laboratory to be released into the venous effluent during hypoxia and are located in subpopulations of coronary vessel endothelial cells in rat heart (Burnstock et al., 1988).
Acknowledgement This work was supported in part by a grant from the British Heart Foundation.
References Berne, R.M., J.M. Gidday, H.E. Hill, R.R. Curnish and R. Rubio, 1987, Adenosine in the local regulation of blood
flow: some controversies, in: Topics and Perspectives in Adenosine Research, eds. E. Gerlach and B.F. Becker (Springer, Berlin) p. 395. Burnstock, G., J. Lincoln, E. Fehrr, A.M. Hopwood, K. Kirkpatrick, P. Milner and V. Ralevic, 1988, Serotonin is localized in endothelial cells of coronary arteries and released during hypoxia: a possible new mechanism for hypoxia-induced vasodilatation of the rat heart, Experientia 44, 705. Busse, R., U. Pohl, C. Kellner and U. Klemm, 1983, Endothelial cells are involved in the vasodilatory response to hypoxia, Pfltigers Arch. 397, 78. Hopwood, A.M. and G. Burnstock, 1987, ATP mediates coronary vasoconstriction via P2x-purinoceptors and coronary vasodilatation via P2v-purinoceptors in the isolated perfused rat heart, European J. Pharmacol. 136, 49. Ishibashi, T., K. Ichihara and Y. Abiko, 1985, Difference in time course between increases in coronary flow and in effluent adenosine concentration during anoxia in the perfused rat heart, Jap. Circ. J. 49, 1090. Moncada, S., R.M.J. Palmer and R.G. Gryglewski, 1986, Mechanism of action of some inhibitors of endothelium-derived relaxing factor, Proc. Natl. Acad. Sci. U.S.A. 83, 9164. Paddle, B.M. and G. Burnstock, 1974, Release of ATP from perfused heart during coronary vasodilatation, Blood Vessels 11,110. Pearson, J.D. and J.L. Gordon, 1979, Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides, Nature 281, 384. ReiUy, W.M., V.L. Saville and G. Burnstock, 1987, An assessment of the antagonistic activity of reactive blue 2 at P1and P2-purinoceptors: supporting evidence for purinergic innervation of the rabbit portal vein, European J. Pharmacol. 140, 47. Sparks, H.V. and M.W. Gorman, 1987, Adenosine in the local regulation of blood flow: current controversies, in: Topics and Perspectives in Adenosine Research, eds. E. Gerlach and B.F. Becker (Springer, Berlin) p. 406.
323
Elsevier EJP 20402 Short communication
Adenosine 5'-triphosphate, adenosine and endothelium-derived relaxing factor in hypoxic vasodilatation of the heart A l e x a n d r a M. H o p w o o d 1, Jill Lincoln, K a r e n A. K i r k p a t r i c k a n d G e o f f r e y B u r n s t o c k * Department of Anatomy and DevelopmentalBiology and Centrefor Neuroscience, University CollegeLondon, Gower Street, London WCIE 6BT, U.K.
Received 20 April 1989, accepted 25 April 1989
The involvement of ATP in hypoxic vasodilatation was investigated using isolated perfused guinea-pig hearts (Langendorff). Reactive blue 2, a selective P2v-purinoceptor antagonist, attenuated dilatations due to ATP and hypoxia. Hydroquinone, an agent which destroys endothelium-derived relaxing factor, substantially decreased dilatations due to 2-methylthioATP, a potent P2v-purinoceptor agonist, and hypoxia, but not to adenosine. ATP may, therefore, have an important role to play in the initiation of hypoxic dilatation which is mediated by the release of endothelium-derived relaxing factor. Hypoxia; Heart; ATP; Adenosine; Endothelium-derived relaxing factor (EDRF)
1. Introduction Since the early 1960s, adenosine has been regarded as the major agent responsible for the vasodilatation seen during hypoxic perfusion (Berne et al., 1987; Sparks and Gorman, 1987). However, methylxanthines, the antagonists of P1purinoceptors are virtually ineffective at reducing the vasodilatation seen during hypoxia or reactive hyperaemia at concentrations where they fully block the dilatations due to adenosine (Sparks and Gorman, 1987). Further, Ishibashi et al. (1985) showed that the time course of adenosine release from hypoxic heart was too late for the elicited vasodilatation to be due to adenosine. In the present study we have tested the hypothesis that ATP is involved in hypoxic vasodilatation by the use of Reactive blue 2 (RB2), a
1 Present address: Syntex Pharmaceuticals Ltd., Syntex House, St. Ives Road, Maidenhead, Berkshire SL6 1RD, U.K. * To whom all correspondence should be addressed.
compound which has recently been shown to be a relatively specific blocker for vasodilatations, including those in coronary vessels, due to ATP, which are mediated via the P2v-purinoceptor (Hopwood and Burnstock, 1987; Reilly et al., 1987). In addition, we have investigated the idea that endothelium-derived relaxing factor (EDRF) is involved in hypoxic vasodilatation by the use of hydroquinone (a free radical quenching agent).
2. Materials and methods Guinea-pigs (250-500 g) of either sex were injected with heparin (2 500 units i.p.) before being killed by cervical dislocation. The heart was removed and cannulated via the aorta for constant flow perfusion (the average flow was 13.6 + 0.3 m l . min -1, n = 28) according to the method of Langendorff. This has been described in detail previously ( H o p w o o d and Burnstock, 1987). Doses of nucleosides and nucleotides were given in 50 /~1 boluses, delivered over 3 s. The order of
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
324
doses of 2-methylthioATP, adenosine and ATP were varied according to Latin square principles. The dose cycle for all these drugs was 8 min. The heart was made hypoxic by switching from Krebs-Henseleit solution (containing (mM): NaC1 115.3, KC1 4.6, MgSO 4 • 7 H 2 0 1.1, N a H C O 3 22.1, KH2PO 4 1.1, CaC12 2.5, glucose 11.1) equilibrated with 95% 02, 5% CO 2 to one equilibrated with 95% N 2 , 5% C O 2. To minimize mixing, the hypoxic Krebs solution was delivered via a second perfusion system (rate-matched pump) which terminated in syringe needle. As this needle was pushed through a self-sealing rubber injection the main pump was switched off using a remote control device (C.E.E.S. Ltd., Essex). This minimized pressure differences and ischaemia likely to occur as a result of such a switchover. The hypoxic perfusion was continued for 1 rain. When the effect of antagonists was examined, control dose-responses to the purines and a control hypoxic dilatation were first obtained and RB2 or hydroquinone were added to the perfusing solution and allowed to act on the heart for 20 rain. The dose-responses and hypoxic dilatations were then repeated in the presence of the antagonist. The results were calculated as the means _+ S.E.M. and the two-tailed Student's t-test was used to assess significance. At the end of the experiment the heart was removed from the cannula, blotted and weighed. The average heart weights were 1.42 _+ 0.04 g (n = 28). Adenosine, ATP (sodium salt), hydroquinone and RB2 were obtained from Sigma. 2-MethylthioATP was obtained from Research Biochemicals Inc. Heparin was obtained from University College Hospital pharmacy.
3. Results
Occasionally, mainly when high concentrations of 2-methylthioATP were applied, biphasic responses were observed in the guinea-pig heart, as previously reported in the rat (Hopwood and Burnstock, 1987). No comment on relative potencies can be made, as responses to ATP and 2methylthioATP were obtained on different preparations.
30. qE 0) ~20.
5
p s S'~ C3
-log moles ATP Fig. 1. The effect of 1.8/xM RB2 on the vasodilatory responses to A T P and and hypoxia. Control dose-response curve to ATP, solid line; + RB2, broken line. The relative antagonism of this response was 15.8, P < 0.01, n = 9. The control hypoxic dilatation is shown by the blank column, and in the presence of RB2 by the dotted column. Hypoxic dilatation was attenuated by 41%, * P < 0.05, n = 9, two-tailed Student's test.
RB2 at 1.8 # M strongly antagonized the vasodilatory responses to ATP; the relative antagonism, taken as the difference between the values of the pD 2 in the absence and presence of RB2, was 15.8 (fig. 1). At this concentration, RB2 also reduced by over 40% the vasodilatation seen during hypoxic perfusion. Both of these effects of RB2 were statistically significant (P < 0.01 and P < 0.05, respectively). No significant effect of RB2 on left ventricular pressure was observed. The mean perfusion pressure of the preparations (69.6 + 5.4 mmHg, n = 9) was not significantly altered by the administration of RB2 (72.6 + 7.1 mmHg, n = 9). Preliminary experiments revealed that, at a concentration of 50 /xM, hydroquinone decreased left ventricular pressure. The present experiments were therefore carded out with hydroquinone at a concentration of 1 /~M where no direct effect on left ventricular pressure was observed. There was no significant difference in the perfusion pressure of the preparations used in the present study, before (67.3 + 4.3 mmHg, n = 6) or after (68.5 _+ 5.1 mmHg, n = 6) hydroquinone. Hydroquinone
325
IE
20.
g
.~ 10 t-1
c~
-log moles 2Me.S.ATP
Adencer~ 1pMo~
Hymxe
Fig. 2. The effect of 1 g M hydroquinone on the vasodilatory responses of guinea-pig heart vasculature to 2-methylthioATP (2.Me.S.ATP), adenosine and hypoxia. Control responses to 2-methylthioATP, solid line; +hydroquinone, broken line. Control responses to adenosine (left-hand bar chart) and hypoxia (right-hand bar chart), blank colunm; +hydroquinone, dotted column. * P < 0.05, * * P < 0.01, * * * P < 0.001, n = 6, two-tailed Student's t-test.
significantly reduced the 2-methylthioATP-mediated dilations but not those elicited by adenosine (fig. 2). The hypoxic dilation was reduced by well over 50%.
4. Discussion
In the present communication we provide pharmacological evidence in support of the view that ATP is involved in eliciting the vasodilatation seen during hypoxic perfusion. We have previously shown that ATP acts at P2v-purinoceptors in the coronary circulation of the rat, distinct from the Pl-purinoceptors at which adenosine exerts its effects (Hopwood and Burnstock, 1987). RB2, a recently proposed P2y-purinoceptor antagonist, was only one-fifth as effective in attenuating the dilatation caused by adenosine as that caused by ATP in the rat heart (Hopwood and Burnstock, 1987) and had no significant effect on adenosine responses in the rabbit portal vein (Reilly et al., 1987). In the present study, RB2 reduced by over 40% the vasodilatation from hypoxic perfusion, which suggests that ATP is involved in the response seen during hypoxia.
The results are consistent with the view that adenosine can no longer be regarded as the major mediator of hypoxic vasodilatation. It is wellestablished that methylxanthines and adenosine deaminase cannot completely abolish the hypoxic vasodilatation (Sparks and Gorman, 1987) and it has recently been reported that adenosine is released too late for it to be able to account for the response seen during the hypoxic perfusion (Ishibashi et al., 1985). Paddle and Burnstock (1974) have provided evidence that ATP may be an alternative mediator of this response, since they noted that ATP was released in the effluent of guinea-pig hearts when these hearts were made hypoxic, in sufficient amounts to be detectable despite its rapid breakdown by ectoenzymes (only about 1% of ATP introduced into the Langendorff heart survived as ATP; the rest was shown to be largely the breakdown product, adenosine). Busse et al. (1983) have found that endothelial cells are important in mediating hypoxic vasodilatation. In these studies, we used hydroquinone, a compound which, by virtue of its free-radical quenching properties, destroys EDRF (Moncada et al., 1986). Our results reveal that this compound significantly reduced the dilatation due to the selective P2y-purinoceptor agonist, 2-methylthioATP, and to hypoxic perfusion, but not the dilatation due to the Pl-purinoceptor agonist, adenosine. This suggests that EDRF is involved in mediating the vasodilatation due to hypoxia. The involvement of EDRF provides indirect evidence that adenosine cannot possibly be the sole mediator of the dilatation seen when the heart is made hypoxic. The source of the ATP leading to vasodilatation in hypoxia has not been established, however, endothelial cells in culture have been shown to release adenine nucleotides (Pearson and Gordon, 1979). In preliminary experiments, attempts to produce a viable Langendorff heart preparation free of endothelium were unsuccessful. Similarly, the guanylate cyclase blocker, methylene blue, proved to be toxic to the preparation. In the absence of data on the effects of other EDRF inhibitors such as haemoglobin, the possibility of RB2 and hydroquinone having effects on other mechanisms, in addition to those already described here in hypoxia, cannot be totally excluded.
326
In conclusion, the pharmacological evidence presented here suggests that ATP has a role in mediating the response to hypoxia. Hypoxic vasodilatation also appears to depend on the release of EDRF, since hydroquinone also reduced the extent of the hypoxic vasodilatation by over 60%. Since only 60% of hypoxic vasodilatation can be abolished by the use of hydroquinone, this implies that only two-thirds of the response is due to endothelial-mediated mechanism Possibilities for the remaining component include locally released agents such as prostanoids and a contribution from vasodilator nerves. Since 40% of the response is blocked by an ATP antagonist, this suggests that ATP is the predominant agent acting via the endothelium. This leaves the question of what agents are responsible for the other 20%. Possibilities include serotonin, acetylcholine and substance P, which have recently been shown in our laboratory to be released into the venous effluent during hypoxia and are located in subpopulations of coronary vessel endothelial cells in rat heart (Burnstock et al., 1988).
Acknowledgement This work was supported in part by a grant from the British Heart Foundation.
References Berne, R.M., J.M. Gidday, H.E. Hill, R.R. Curnish and R. Rubio, 1987, Adenosine in the local regulation of blood
flow: some controversies, in: Topics and Perspectives in Adenosine Research, eds. E. Gerlach and B.F. Becker (Springer, Berlin) p. 395. Burnstock, G., J. Lincoln, E. Fehrr, A.M. Hopwood, K. Kirkpatrick, P. Milner and V. Ralevic, 1988, Serotonin is localized in endothelial cells of coronary arteries and released during hypoxia: a possible new mechanism for hypoxia-induced vasodilatation of the rat heart, Experientia 44, 705. Busse, R., U. Pohl, C. Kellner and U. Klemm, 1983, Endothelial cells are involved in the vasodilatory response to hypoxia, Pfltigers Arch. 397, 78. Hopwood, A.M. and G. Burnstock, 1987, ATP mediates coronary vasoconstriction via P2x-purinoceptors and coronary vasodilatation via P2v-purinoceptors in the isolated perfused rat heart, European J. Pharmacol. 136, 49. Ishibashi, T., K. Ichihara and Y. Abiko, 1985, Difference in time course between increases in coronary flow and in effluent adenosine concentration during anoxia in the perfused rat heart, Jap. Circ. J. 49, 1090. Moncada, S., R.M.J. Palmer and R.G. Gryglewski, 1986, Mechanism of action of some inhibitors of endothelium-derived relaxing factor, Proc. Natl. Acad. Sci. U.S.A. 83, 9164. Paddle, B.M. and G. Burnstock, 1974, Release of ATP from perfused heart during coronary vasodilatation, Blood Vessels 11,110. Pearson, J.D. and J.L. Gordon, 1979, Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides, Nature 281, 384. ReiUy, W.M., V.L. Saville and G. Burnstock, 1987, An assessment of the antagonistic activity of reactive blue 2 at P1and P2-purinoceptors: supporting evidence for purinergic innervation of the rabbit portal vein, European J. Pharmacol. 140, 47. Sparks, H.V. and M.W. Gorman, 1987, Adenosine in the local regulation of blood flow: current controversies, in: Topics and Perspectives in Adenosine Research, eds. E. Gerlach and B.F. Becker (Springer, Berlin) p. 406.