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Diadenosine triphosphate (Ap3A) is a vasoactive mediator stored in platelet granules that may be released during coronary ischemia-reperfusion. To study its coronary effects in such circumstances, rat hearts were perfused in a Langendorff preparation and the coronary response to Ap3A (10(-7)-10(-5) mol/L) was recorded. Both at basal coronary resting tone and after precontraction with 11-dideoxy-1a,9a-epoxymethanoprostaglandin F(2)(α) (U46619), Ap3A produced concentration-dependent vasodilation in the heart, which was attenuated following ischemia-reperfusion. Ap3A-induced relaxation was also attenuated in control conditions and after ischemia-reperfusion by the purinergic P2Y antagonist reactive blue 2 (2 × 10(-6) mol/L), the P2Y(1) antagonist MRS 2179 (10(-5) mol/L), the nitric oxide synthesis inhibitor N-omega-nitro-l-arginine methyl ester (l-NAME; 10(-4) mol/L) and the ATP-dependent potassium channel blocker glibenclamide (10(-5) mol/L). These results suggest that Ap3A induces coronary vasodilation, an effect attenuated by ischemia-reperfusion due to the functional impairment of purinergic P2Y receptors and K(ATP) channels, and/or reduced nitric oxide release. This impairment of vasodilation may contribute to the coronary dysregulation that occurs during ischemia-reperfusion.

Diadenosine tetraphosphate (AP4A) is a vasoactive mediator that may be released from platelet granules and that may reach higher plasma concentrations during coronary ischemia-reperfusion. The objective of this study was to analyze its coronary effects in such conditions. To this, rat hearts were perfused in a Langendorff preparation and the coronary response to Ap4A (10(-7)-10(-5) M) was recorded. In control hearts, Ap4A produced concentration-dependent vasodilatation both at the basal coronary resting tone and after precontracting coronary vasculature with 11-dideoxy-1a,9a-epoxymethanoprostaglandin F2α (U46619), and this vasodilatation was reduced by reactive blue 2 (2×10(-6) M), glibenclamide (10(-5) M), H89 (10(-6) M), U73122 (5×10(-6) M) and endothelin-1 (10(-9) M), but not by L-NAME (10(-4) M), isatin (10(-4) M), GF109203x (5×10(-7) M), or wortmannin (5×10(-7) M). After ischemia-reperfusion, the vasodilatation to Ap4A diminished, both in hearts with basal or increased vascular tone, and in this case the relaxation to Ap4A was not modified by reactive blue 2, L-NAME, glibenclamide, isatin, H89, GF109203x or wortmannin, although it was reduced by U73122 and endothelin-1. UTP produced coronary relaxation that was also reduced after ischemia-reperfusion. These results suggest that the coronary relaxation to Ap4A is reduced after ischemia-reperfusion, and that this reduction may be due to impaired effects of KATP channels and to reduced response of purinergic P2Y receptors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To analyze the effects of endothelin-1 on the b-adrenergic response of the coronary circulation, 2-mm-long segments of coronary arteries from rats were prepared for isometric tension recording in organ baths. The relaxation to isoproterenol (3 x 10(-8) M), field electrical stimulation (4 Hz, 0.1-millisecond duration, 10 seconds), acetylcholine (3 x 10(-8) M), and sodium nitroprusside (10(-9) M) was recorded in arteries precontracted with U46619 (10(-7) to 5 x 10(-7) M) before and after treatment with endothelin-1 (3 3 10210 and 1029 M). The relaxation to isoproterenol was increased by treatment with endothelin-1 and with the endothelin ET(B) antagonist BQ788 (10(-6) M) but not with the endothelin ET(A) antagonist BQ123 (10(-6) M) or with the blocker of protein kinase C chelerythrine (10(-5) M). In the presence of BQ788, BQ123, or chelerythrine, endothelin-1 did not modify the relaxation to isoproterenol. Treatment with endothelin-1 did not modify the relaxation to electrical stimulation, acetylcholine, or sodium nitroprusside. These results suggest that endothelin-1 may potentiate coronary beta-adrenergic vasodilatation, at least in part due to stimulation of endothelin ET(A) receptors and activation of protein kinase C.

Aims: Diadenosine polyphosphates are vasoactive mediators that may be released from platelet granules and which may be present at higher concentrations during coronary ischaemia-reperfusion. The objective of this study was to analyse their effects in such conditions., Methods and Results: Rat hearts were perfused in a Langendorff preparation and the response to diadenosine pentaphosphate (Ap5A, 10(-7)-10(-5) M) was recorded. In control hearts, Ap5A produced a small, transient coronary vasoconstriction followed by marked vasodilatation, as well as a reduction in the left ventricular developed pressure dP/dt and heart rate, both at the basal coronary resting tone or after pre-contracting coronary arteries with 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F2alpha (U46619). After ischaemia-reperfusion, the vasoconstriction in response to Ap5A was augmented and vasodilatation diminished, both in hearts with basal or increased vascular tone. The pyridoxal derivative P(2) purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 3 x 10(-6) M), inhibited this vasoconstriction, while the antagonist of purinergic P(2Y) receptors, Reactive Blue 2 (2 x 10(-6) M), inhibited the vasodilatation, both before and after ischaemia-reperfusion. The antagonist of nitric oxide synthesis N-omega-nitro-L- arginine methyl ester (L-NAME, 10(-4) M) did not modify the response to Ap5A, whereas the cyclooxygenase inhibitor, meclofenamate (2 x 10(-6) M), reduced contraction and increased the relaxation in response to Ap5A after ischaemia-reperfusion but not under control conditions., Conclusion: Ischaemia-reperfusion reduces the vasodilatory response to Ap5A and increases the vasoconstriction provoked due to a reduced influence of purinergic P(2Y) receptors and/or to the production of vasoconstrictor prostanoids.

Hearts from Sprague-Dawley rats were perfused at constant flow and then exposed to 30 min global zero-flow ischemia followed by 15 min reperfusion. After ischemia-reperfusion, coronary arteries were dissected from the heart and segments 2 mm long were prepared for isometric tension recording in organ baths. Stimulation of the arteries with 5-hydroxytryptamine (10(-6) M) produced contraction, which was potentiated by treatment with endothelin-1 (3x10(-10); 10(-9) M). This potentiation was lower in the arteries from hearts after ischemia-reperfusion (for 3x10(-10) M, 15+/-5%; P>0.05; for 10(-9) M, 37+/-7%, P<0.01, n=5) than after control (for 3x10(-10) M, 34+/-4%; P<0.01; for 10(-9) M, 50+/-6%, P<0.01, n=5), and the potentiation was reduced by the inhibitor of nitric oxide synthesis l-NAME (10(-4) M), the antagonist of endothelin ET(A) receptors BQ123 (10(-6) M) and the antagonist of endothelin ET(B) receptors BQ788 (10(-6) M), but not by the cyclooxygenase inhibitor meclofenamate (10(-5) M). These results suggest that endothelin-1 at low concentrations potentiates coronary vasoconstriction, and this effect is reduced after ischemia-reperfusion, mediated by endothelin ET(A) and ET(B) receptors and dependent on nitric oxide release.

To study the effects of arterial pressure on coronary reactive hyperaemia, left circumflex coronary artery flow was measured, and reactive hyperaemia was determined after 5, 10 or 20 s of occlusion of this artery in anaesthetized goats during normotension, hypertension and hypotension. During hypertension induced by aortic constriction (mean arterial pressure, MAP = 140 +/- 6 mmHg) coronary vascular resistance (CVR), reactive hyperaemia (ratio of peak in hyperaemic flow to control flow and ratio of repayment to debt) and the decrease in CVR during the peak in hyperaemic flow were comparable to those during normotension. During hypertension induced by noradrenaline (MAP = 144 +/- 6 mmHg) CVR was 16% lower (P < 0.05), reactive hyperaemia was reduced by 14-25% (P < 0.05) and the decrease in CVR during the peak in hyperaemic flow was lower than the values of these parameters during normotension. During hypotension induced by constriction of the caudal vena cava (MAP = 40 +/- 4 mmHg) CVR was 22% lower (P < 0.05), reactive hyperaemia was reduced by 25-65% (P < 0.05) and the decrease in CVR during the peak in hyperaemic flow was less compared to the values of these parameters during normotension. During hypotension induced by isoprenaline (MAP = 45 +/- 4 mmHg) CVR was 59% lower, reactive hyperaemia was reduced by 55-100% (P < 0.01) and the decrease in CVR during the peak in hyperaemic flow was less compared to the values of these parameters during normotension. Arterial pressure is a main determinant of coronary reactive hyperaemia after brief periods of ischaemia, and the relationship between arterial pressure and reactive hyperaemia may depend in part on changes in CVR after variations in arterial pressure. These changes in CVR may be related to the action on coronary vessels of myocardial factors and vascular myogenic mechanisms.

To compare the effects of antagonists for endothelin ET(A) and ET(B) receptors on the action of ischemia-reperfusion on endothelial and myocardial function, 30 min of partial or total occlusion followed by 60 min of reperfusion of the left circumflex coronary artery was induced in anesthetized goats treated with intracoronary administration of saline (vehicle), BQ-123 (endothelin ET(A) receptors antagonist) or BQ-788 (endothelin ET(B) receptors antagonist). During reperfusion after partial occlusion, coronary vascular conductance and left ventricle dP/dt were decreased after saline or BQ-788, and they normalized after BQ-123. In these three groups of animals, the coronary effects of acetylcholine (3-100 ng) and sodium nitroprusside (1-10 microg) during reperfusion were as under control. During reperfusion after total occlusion, coronary vascular conductance and left ventricle dP/dt were decreased after saline, and they normalized after BQ-123 or BQ-788. In these three groups of animals, the coronary effects of acetylcholine but not those of sodium nitroprusside during reperfusion were decreased after saline, and they reversed after BQ-123 or BQ-788. Therefore, selective antagonists of endothelin ET(B) and ET(A) receptors may produce similar protection of coronary vasculature and myocardium against reperfusion after severe ischemia. Selective antagonists of endothelin ET(B) receptors, contrarily to those of endothelin ET(A) receptors, may be ineffective to protect coronary vasculature and myocardium against reperfusion after mild ischemia.

To analyse the coronary effects of endothelin-1 after ischemia-reperfusion, the left anterior descending coronary artery of anesthetized pigs was subjected to 30-min occlusion followed by 60-min reperfusion. Then, rings distal (ischemic arteries) and proximal (control arteries) to the occlusion were taken from this artery and prepared for isometric tension recording. The sensitivity of the contraction in response to endothelin-1 (3 x 10(-10)-3 x 10(-7) M) and the endothelin ET(B) receptor agonist IRL-1620 (3 x 10(-10)-3 x 10(-7) M) was greater in ischemic vessels. The endothelin ET(A) receptor antagonist BQ-123 (10(-7)-3 x 10(-6) M) decreased the sensitivity of the response to endothelin-1 similarly in ischemic and control arteries. The endothelin ET(B) receptor antagonist BQ-788 (10(-6) M), endothelium removal or the inhibitor of nitric oxide synthesis N(omega)-nitro-L-arginine methyl ester (L-NAME 10(-4) M) potentiated the response to endothelin-1 and IRL-1620 in control arteries only. The cyclooxygenase inhibitor meclofenamate (10(-5) M) augmented the maximal response to endothelin-1 in control arteries, and reduced it in ischemic arteries. In precontracted arteries, IRL-1620 (3 x 10(-11)-3 x 10(-10) M) relaxed control but not ischemic arteries, and L-NAME or meclofenamate abolished this relaxation. Therefore, ischemia-reperfusion increases the coronary vasoconstriction in response to endothelin-1 probably due to impairment of endothelin ET(B) receptor-induced release of nitric oxide and prostacyclin, augmentation of the contractile response to activation of endothelin ET(B) receptors, and increased release of vasoconstrictor prostanoids.

Urocortin is a vasodilator peptide related to corticotrophin-releasing factor, which may protect myocardium during coronary ischemia-reperfusion. To study whether urocortin also protects coronary endothelial function during ischemia-reperfusion, hearts from Sprague-Dawley rats were perfused at constant flow and then exposed to 15 mins ischemia followed by 15 mins reperfusion. In one series of experiments, we found that the coronary relaxation to urocortin (10(-11) to 10(-8) M) was reduced by ischemia-reperfusion (51 +/- 4% vs. 79 +/- 4% of the active tone, for the 10(-10) Mdose). In other series of experiments, we observed that ischemia-reperfusion reduced the coronary relaxation to a test dose of acetylcholine (10(-6) M) (25 +/- 2% vs. 54 +/- 9% of active tone), without modifying the relaxation to sodium nitroprusside (10(-6) M). Treatment with a low threshold concentration of urocortin (10(-11) M), administered before ischemia and during reperfusion, partly improved the coronary relaxation to acetylcholine (36 +/- 3% of active tone). These results suggest that ischemia-reperfusion impairs the coronary vasodilation to urocortin and produces endothelial dysfunction and that this endothelial dysfunction may be improved by urocortin.

To compare the coronary effects of endothelin-1 and vasopressin, as well as the role of nitric oxide (NO) and prostanoids in these effects, blood flow in the left circumflex (73 animals) or left anterior descending (19 animals) coronary artery (coronary flow) was electromagnetically measured, and both peptides were intracoronarily injected in anesthetized goats under control conditions and after intravenous administration of the inhibitor of NO synthesis NW-nitro-L-arginine methyl ester (L-NAME, 47 mg/kg, nine animals) or the inhibitor of cyclooxygenase meclofenamate (6-8 mg/kg, seven animals). In every animal, both endothelin-1 and vasopressin reduced coronary flow in a dose-dependent way, but these reductions by 0.03, 0.1 and 0.3 nmol of endothelin-1 (16%, 33% and 66%, respectively) were significantly higher than those by the equimolar doses of vasopressin (11%, 22% and 35%, respectively). After L-NAME treatment, the reductions of coronary flow by both peptides were augmented, and this augmentation was about two times higher for endothelin-1 than for vasopressin. Meclofenamate treatment did not affect the reductions of coronary flow caused by both peptides. Therefore, we suggest that endothelin-1 is more effective than vasopressin for producing coronary vasoconstriction, but vasopressin also produces remarkable coronary vasoconstriction. Also, it is suggested that NO may play a more relevant role for modulating the coronary vasoconstriction by endothelin-1 than by vasopressin, and that cyclooxygenase products may not be involved in the coronary effects of these two peptides.

To examine the effects of reperfusion after short and prolonged ischemia on the coronary action of endothelin-1, left circumflex coronary artery flow was electromagnetically measured, and 15- or 60-min occlusion of this artery followed by reperfusion was induced in anesthetized goats. In non-treated animals, during reperfusion after 15-min occlusion, the duration but not the peak of endothelin-1-induced coronary effects (0.01-0.3 nmol) was increased, and the effects of acetylcholine (3-100 ng) were unchanged. During reperfusion after 60-min occlusion, the peak and duration of endothelin-1-induced effects were increased whereas those of acetylcholine were decreased. N(w)-nitro-L-arginine methyl esther (L-NAME) treatment did not modify the peak and duration of the coronary effects of endothelin-1 during reperfusion after both durations of occlusion. This treatment inhibited the effects of the two higher doses but not those of the two lower doses of acetylcholine during reperfusion after 15-min occlusion, and it did not modify the effects of any dose of this drug during reperfusion after 60-min occlusion. Meclofenamate treatment did not modify the coronary effects of endothelin-1 and acetylcholine during reperfusion after both durations of occlusion. These results suggest that ischemia-reperfusion increases the coronary response to endothelin-1, which is more pronounced during reperfusion after prolonged than after brief ischemia, and that this increased response is probably related to inhibition of nitric oxide release, without involvement of prostanoids.

To study the role of K(+) channels in the coronary and renal vascular response to vasopressin during diabetes mellitus, and whether there are gender differences in this role, we have examined the isometric response to this peptide of 2-mm-long arterial segments from male and female, normoglycemic and streptozotocin-induced diabetic rats. Vasopressin (10(-12)-3 x 10(-8) M) produced arterial concentration-dependent contraction, and during normoglycemia, this contraction was lower in coronary arteries from female than from male rats, and it was similar in renal arteries from both genders. This contraction was reduced by diabetes in coronary arteries, and increased in renal arteries, from both genders. The blocker of Ca(2+)-sensitive K(+) channels charybdotoxin (10(-7) M) increased the contraction to vasopressin in coronary arteries of diabetic females, but not in the other cases (diabetic males and normoglycemic females or males). This blocker also increased the contraction to vasopressin in renal arteries from diabetic, but not in those from normoglycemic female rats, and also increased it in a higher magnitude in arteries from diabetic than in those from normoglycemic male rats. The blocker of ATP-sensitive K(+) channels glybenclamide (10(-5) M) or the scavenger of superoxide radicals superoxide dismutase (100 U/ml) did not modify the contraction to vasopressin in any experimental group. These results suggest that diabetes activates the modulatory role of K(+) channels in the coronary and renal vasoconstriction to vasopressin, but it alters in a different way the vasoconstriction to vasopressin in these two types of arteries. The effects of diabetes on this vasoconstriction are not related to increased release of superoxide radicals.

To examine coronary reactivity to acetylcholine and endothelin-1 (ET-1) during partial ischaemia and reperfusion, flow in the left circumflex coronary artery was measured electromagnetically, and coronary partial ischaemia was induced by stenosis of this artery in anaesthetized goats. In eight animals not treated with N(G)-nitro-l-arginine methyl ester (l-NAME), coronary stenosis reduced coronary flow by 45%, mean arterial pressure by 16% and coronary vascular conductance by 34%. During this ischaemia, coronary vasodilatation to acetylcholine (0.003-0.1 microg) and sodium nitroprusside (SNP; 1-10 microg) was markedly reduced, and coronary vasoconstriction to ET-1 (0.01-0.3 nmol) was attenuated. After 30 min of reperfusion, coronary flow, mean arterial pressure and coronary vascular conductance remained decreased, and the effects of acetylcholine, SNP and ET-1 were as in control animals. In six goats treated with N(G)-nitro-l-arginine methyl ester, coronary stenosis reduced coronary flow by 26% and coronary vascular conductance by 24%, but did not affect mean arterial pressure. During this ischaemia, coronary vasodilatation to acetylcholine and SNP was also markedly reduced, but vasoconstriction to ET-1 was unaffected. After 30 min of reperfusion, coronary flow and coronary vascular conductance remained decreased and mean arterial pressure was normal; in addition, the effects of acetylcholine were lower, those of SNP were similar and those of ET-1 were higher than in control animals. Therefore partial ischaemia reduces the coronary vasodilator reserve and blunts coronary vasoconstriction to ET-1, and reperfusion does not alter the endothelium-dependent and -independent coronary vasodilatation or vasoconstriction to ET-1.

To examine the role of vasopressin V1 and V2 receptors, nitric oxide and prostanoids in the coronary vascular effects of [Arg8]vasopressin, coronary blood flow was measured with an electromagnetic flow transducer placed around the left circumflex (23 goats) or anterior descending (11 goats) coronary artery and vasopressin (0.03-1 microg) was intracoronarily injected in 34 anesthetized, open-chest goats. Basal mean values for coronary blood flow, mean systemic arterial pressure and heart rate, were 34 +/- 2.38 ml/min, 89 +/- 3.34 mmHg and 80 +/- 3.06 beats/min, respectively. Vasopressin produced dose-dependent decreases in coronary blood flow and the maximal reduction of this flow, attained with 1 microg of vasopressin, was 14 +/- 1.49 ml/min (42 +/- 2.64% of basal flow) (P < 0.01). Desmopressin (0.03-1 microg; 8 goats) did not affect significantly coronary blood flow. The intracoronary infusion of the antagonist for vasopressin V1 receptors d(CH2)5Tyr (Me) arginine vasopressin (2 microg/min per kg, 6 animals) significantly diminished the effects of vasopressin on coronary blood flow (the effects of 1 microg of vasopressin were reduced by 28%, P < 0.05). The mixed antagonist for vasopressin V1 and V2 receptors desGly-d(CH2)5-D-Tyr(Et)Val arginine vasopressin (0.2, 0.7 and 2 microg/min per kg, 9 animals) decreased in a dose-dependent manner the effects of vasopressin on coronary blood flow (the effects of 1 microg of vasopressin were decreased by 61% with 2 microg/min per kg, P < 0.01). Intracoronary infusion of saline (vehicle, 3 goats) did not change the effects of vasopressin on coronary blood flow. Intravenous administration of the inhibitor of nitric oxide synthesis N-omega-nitro-L-arginine methyl ester (L-NAME, 47 mg/kg, 9 animals) decreased resting coronary blood flow by 10% (P < 0.01) and augmented mean systemic arterial pressure by 20% (P < 0.01), without changing heart rate. During this treatment the reduction in coronary blood flow produced by vasopressin was higher than under control (the effects of 1 microg of vasopressin were increased by 28%, P < 0.01). Intravenous administration of the inhibitor of cyclooxygenase, meclofenamate (5 mg/kg, 7 animals), neither modified resting coronary blood flow, arterial pressure and heart rate nor the effects of vasopressin on this flow. These data indicate that vasopressin produces marked coronary vasoconstriction and suggest that: (a) it may be mediated by vasopressin V1 receptors, without involvement of vasopressin V2 receptors, (b) it is probably inhibited by nitric oxide under normal conditions and (c) it may be not modulated by prostanoids.

Endothelin-1 (ET-1) effects on left circumflex coronary artery (LCCA) flow, systemic arterial pressure, heart rate, and left ventricle (LV) pressure and dP/dt were recorded in five anesthetized goats under control conditions and during arterial hypotension. Hypotension, induced by mechanical constriction of the inferior vena cava, reduced mean systemic arterial pressure from 92 +/- 4 to 59 +/- 4 mm Hg (p < 0.002), LCCA flow from 26 +/- 4 to 17 +/- 3 ml/min (p < 0.01), systolic LV pressure from 108 +/- 2.5 to 79 +/- 2 mm Hg (p < 0.002), and peak systolic LV dP/dt from 1,500 +/- 106 to 990 +2- 130 mm Hg/s (p < 0.02). Heart rate did not change (p > 0.05). ET-1 (0.01-0.1 nmol) injected into LCCA reduced LCCA flow in a dose-dependent manner without affecting the other variables recorded during control periods (C) and hypotension (H). The percentage reductions of LCCA flow by ET-1 were: for 0.01 nmol, 4.5 +/- 2 (C) vs. 11 +/- 2 (H) (p < 0.05); for 0.03 nmol, 15 +/- 2 (C) vs. 32 +/- 4 (H) (p < 0.05); and for 0.1 nmol, 44 +/- 5 (C) vs. 67 +/- 5 (H) (p < 0.05). This indicates that the coronary vasoconstrictor effects of ET-1 are increased during hypotension. Therefore, heart ischemia may be aggravated in situations of arterial hypotension with increased plasma concentrations of ET-1.