Ronald J. Shebuski

Prevention of blockage of partially obstructed coronary arteries with prostacyclin correlates with inhibition of platelet aggregation

Coronary arteries (circumflex or left anterior descending) of anesthetized dogs were partially obstructed to approximately 5% of the normal lumen size by fitting a plastic cylinder around the vessel. Under these conditions, blood flow in the artery was not maintained but, instead, gradually declined over a few minutes until the vessel was completely blocked. Shaking the plastic obstructor restored blood flow temporarily, however, flow gradually declined again to zero. Sometimes flow was spontaneously restored by immediate increases that occurred at irregular intervals while, on other occasions, blood flow had to be restored by shaking the obstructor every time the rate declined to near zero. Intravenous infusion of prostacyclin (PGI2) at 15 to 150 ng/kg/min reversed and prevented the blockage of the coronary arteries. The efficacy of PGI2 in preventing blockage correlated with inhibition of ADP-induced platelet aggregation in platelet rich plasma prepared from blood samples withdrawn from the dogs during PGI2 infusion. Other coronary vasodilators, nitroglycerin and PGE2, that have no antiaggregatory effects, failed to prevent blockage whereas PGE1 and indomethacin, which do block aggregation, also prevented blockage of the vessels. PGI2 or its precursor, PGH2, dripped topically on the obstructed site prevented the blockage of the artery. This local effect of IGI2 could be obtained with amounts too small to cause systemic inhibition of platelet aggregation. The results show that PGI2 prevents blockage of partially obstructed coronary arteries and this effect correlates with inhibition of platelet aggregation. Furthermore, the data suggest that locally produced PGI2 may have a local antiaggregatory effect without inhibiting platelet aggregation in the general circulation.

Reduction of myocardial infarct size by ramiprilat is independent of angiotensin II synthesis inhibition

The angiotensin-converting enzyme inhibitor ramiprilat, the angiotensin II receptor antagonist losartan, angiotensin II, ramiprilat plus angiotensin II, or saline (N = 6 each group), were administered i.v. in anesthetized, open-chest rabbit preparations of acute myocardial ischemia. Animals were instrumented for measurement of systemic hemodynamics and left ventricular +dP/dtmax, then subjected to 30 min of left anterior descending coronary artery occlusion (marginal branch) followed by 2 h of reperfusion. Ramiprilat (50 micrograms/kg), losartan (10 mg/kg), or saline were administered prior to reperfusion, and angiotensin II (2.5 ng/kg per min) was infused 15 min prior to occlusion and throughout the remainder of the experiment. Losartan was supplemented (10 mg/kg) after 1 h of reperfusion. These non-hypotensive doses of ramiprilat and losartan were demonstrated to significantly antagonize the systemic pressor effects of i.v. challenge with angiotensin I (15% of control, maximum) and II (5% of control, maximum), respectively, for the duration of the experiment. Systemic hemodynamic and +dP/dtmax changes due to occlusion/reperfusion or drug administration were similar between treatment groups. Infarct size was measured post-experimentally using tetrazolium staining and is reported as a percent of area at risk. Infarct size/area at risk (%) was significantly lower in rabbits administered ramiprilat only (20 +/- 6%*) or ramiprilat plus angiotensin II (26 +/- 5%*), compared to those receiving saline (41 +/- 6%), angiotensin II (51 +/- 4%), or losartan (52 +/- 4%, mean +/- S.E.M., * P < 0.05). These data indicate that direct angiotensin II receptor stimulation or receptor antagonism does not alter the degree of myocardial necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ischemic preconditioning fails to limit infarct size in reserpinized rabbit myocardium. Implication of norepinephrine release in the preconditioning effect.

BackgroundInfarct size reduction by ischemic preconditioning is believed to be mediated by adenosine; however, whether adenosine is the factor responsible for the initiation of this protection remains unknown. It is possible that during preconditioning, adenosine stimulates receptors on presynaptic nerve terminals and retards the release of norepinephrine (NE) during the prolonged ischemia or that NE release during preconditioning augments adenosine production. Methods and ResultsTo test whether the release of NE is involved in the preconditioning phenomenon, rabbits were pretreated with reserpine (5 mg/kg sc, 24 hours before) to deplete presynaptic nerve terminals of NE stores. On the day of the experiment, the rabbits were anesthetized with ketaminexylazine and instrumented for coronary occlusion. Nonreserpinized animals were used as controls. The control group (n=7) was subjected to 30 minutes of coronary occlusion and 120 minutes of reperfusion (ischemia-reperfusion) only. The preconditioned group (n=10) received 5 minutes of preconditioning ischemia and 10 minutes of reperfusion before the prolonged ischemia-reperfusion. Of the reserpinized animals, half (n=7) received preconditioning before ischemia-reperfusion and the remaining animals (n=7) did not. At termination of the experiment, an intravenous tyramine challenge (1 mg/kg) was used to confirm NE depletion in reserpinized rabbits. The resulting infarcts were measured with tetrazolium and planimetry. With comparable hemodynamics and areas at risk, infarct size in control animals was 39.8±2.1% of the risk region. Preconditioned animals showed an expected reduction of infarct size to 14.8±2.2% of risk region (P<.05 vs control). Of the reserpinized animals, those that received reserpine alone had infarcts that were 38.5±4.5% of risk region, and those that were preconditioned had infarcts that were 41.4±3.6% of risk region, which was not significantly different than the control group. ConclusionsWe conclude that preconditioning fails to protect ischemic-reperfused myocardium in reserpinized rabbit myocardium, indicating that the release ofNE during either preconditioning or prolonged ischemia is critical to preconditioning mediated protection.

Comparison in anesthetized dogs of the anti-aggregatory and hemodynamic effects of prostacyclin and a chemically stable prostacyclin analog, 6a-carba-PGI2 (carbacyclin)

The intravascular anti-aggregatory and systemic and hemodynamic effects of prostacyclin and carbacyclin were compared by intravenous infusion in pentabarbital anesthetized dogs. Ten times as much carbacyclin was needed to produce comparable inhibition of platelet aggregation in the lumen of partially obstructed circumflex coronary arteries. These doses of carbacyclin caused similar decreases in total peripheral resistance as equi-effective anti-aggregatory doses of prostacyclin. There was a trend for the decrease in blood pressure with carbacyclin to be less than that produced by equi-effective anti-aggregatory doses of prostacyclin because carbacyclin caused somewhat greater increases in cardiac output. Changes in heart rate were similar with both substances. During carbacyclin and prostacyclin infusion resistance in normal (unobstructed) coronary arteries decreased. Both substances had comparable effects on pulmonary vascular resistance, right atrial pressure and left ventricular dp/dt at equivalent anti-aggregatory doses both before and after atropine (1 mg/kg) and hexamethonium (5 mg/kg). During 5 to 6 hour infusions of carbacyclin there was no evidence of desensitization of dog platelets to the anti-aggregatory activity. These results show that carbacyclin has a similar spectrum of activity as prostacyclin and is about one-tenth as potent.

Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1

OBJECTIVE A blocking monoclonal antibody to intercellular adhesion molecule-1 (ICAM-1), CL18/6, previously has been demonstrated to inhibit neutrophil attachment to isolated vascular endothelium and cardiomyocytes. Due to the well known participation of ICAM-1 in the inflammatory responses associated with myocardial ischemia/reperfusion injury, we investigated if CL18/6 could attenuate myocardial ischemia/reperfusion injury in vivo. METHODS Saline (3-5 ml, i.v., n = 6), non-blocking control MAb CL18/1D8 or CL18/6 (both 0.5 mg kg-1, i.v., n = 4) were administered prior to coronary occlusion (1 h) and subsequent reperfusion (5 h) produced by inflation of a coronary balloon angioplasty catheter in isoflurane-anesthetized, closed-chest dogs. Heart rate and arterial pressure were measured, and regional myocardial blood flow (rMBF), and myeloperoxidase activity (MPO) to index local neutrophil sequestration, were determined. Myocardial infarct size (IS) was evaluated using the tetrazolium staining technique and expressed as a percent of area at risk (AR). RESULTS Changes in heart rate and arterial pressure were insignificant throughout the experiment. rMBF (mean +/- s.e.m.) in the ischemic subendocardium for each treatment group was: Saline (0.07 +/- 0.02 ml min-1 g-1); CL18/1D8 (0.04 +/- 0.02); CL18/6 (0.06 +/- 0.02). IS/AR% was: saline (37 +/- 3%); CL18/1D8 (39 +/- 9%); CL18/6 (15 +/- 4%*); * = significantly different from CL18/1D8 and saline, P < 0.05. MPO assayed from AR immediately adjacent to the infarct was significantly reduced below infarct MPO only in the CL18/6 treated group-36%). CONCLUSIONS The results indicate that CL18/6 antagonism of ICAM-1 provided cardioprotection associated with reduced neutrophil activity in vulnerable myocardium, and suggest that ICAM-1 mediated neutrophil sequestration in endangered cardiac tissue is an important mechanism of myocardial ischemia/reperfusion injury.

6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.

Prevention of Reocclusion following Tissue-Type Plasminogen Activator-Induced Thrombolysis by the RGD-Containing Peptide, Echistatin, in a Canine Model of Coronary Thrombosis

We evaluated the effect of the RGD-containing peptide, echistatin, on thrombolysis time and acute reocclusion in a canine model of coronary thrombosis/thrombolysis. Occlusive thrombus formation was induced by electrical injury, via a stimulating electrode, to the endothelial surface of the circumflex coronary artery in the open-chest, anesthetized dog in the presence of a critical stenosis. Fifteen minutes after occlusive thrombus formation, dogs received either an intravenous infusion of vehicle (saline at 0.1 ml/min) or echistatin (15 micrograms/kg/min i.v.). Heparin was given as an initial bolus (100 U/kg i.v.) 15 min after thrombus formation and repeated at hourly intervals (50 U/kg). This dose of heparin increased activated partial thromboplastin time to 1.5- to 2.5- fold over control. Thrombolysis was induced with recombinant tissue-type plasminogen activator (tPA) at a total dose of 1 mg/kg, intravenously administered over 90 min with 10% given as an initial bolus. The vehicle-treated animals reperfused at 48 +/- 9 min with a reperfusion incidence of 60% (3/5). The echistatin-treated animals reperfused at 46 +/- 5 min with a reperfusion incidence of 100% (5/5). After stopping the tPA infusion, acute reocclusion occurred in 100% (3/3) of the vehicle-treated dogs and in only 20% (1/5) of the echistatin-treated dogs. Echistatin caused a greater than 5-fold increase in buccal mucosa bleeding time and almost completely inhibited ex vivo platelet aggregation to ADP, collagen, and U-46619. Residual thrombus wet weight, determined at the end of the experiment, was significantly lower for the echistatin group (2.1 +/- 0.2 mg) compared to the vehicle group (5.8 +/- 0.7 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative Effects of the Potassium Channel Openers Cromakalim and Pinacidil and the Cromakalim Analog U-89232 on Isolated Vascular and Cardiac Tissue

ATP-sensitive potassium (K+ATP) channel openers such as cromakalim and pinacidil exhibit both potent vasodilatory and anti-ischemic properties. U-89232, a cyanoguanidine analog of cromakalim, has recently been found to exhibit myocardial protection during ischemia without altering in vivo hemodynamics. We examined the effects of U-89232, cromakalim and pinacidil in isolated vascular and cardiac tissue and tested whether glyburide, a KATP channel blocker, could antagonize their effects. All three compounds produced concentration-dependent relaxation in isolated vascular segments, with cromakalim being approximately 100-fold more potent than either pinacidil or U-89232. Glyburide completely antagonized the effects of pinacidil but merely blunted the effects of cromakalim and U-89232. In an isolated rabbit cardiac tissue preparation, U-89232 had little effect on maximum tension in cardiac muscle, whereas cromakalim and pinacidil significantly decreased maximum developed tension in a concentration-dependent manner. Glyburide effectively antagonized the effects of cromakalim and pinacidil in cardiac tissue. These data suggest that U-89232, although chemically related to cromakalim, possesses activity which is not common to known potassium channel openers.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF A POTENT ANTIPLATELET 7-AMINOFUROCHROMONE

Abstract The synthesis of a series of rigid analogs designed to explore the conformational requirements associated with the related 2-aminochromone 1 led to the identification of class of novel antiplatelet 7-aminofurochromes 2 . A potent derivative 13 was further evaluated with regard to mechanism of action as well as in a canine model of platelet dependent thrombus formation.

Interaction of Dopamine, (±)-Dobutamine and the (−)-Enantiomer of Dobutamine with α- and β-Adrenoceptors in the Pulmonary Circulation of the Dog

The effects of dopamine, (±)-dobutamine (racemic mixture) and (–)-dobutamine on α-adrenoceptor-mediated vasoconstriction were evaluated in the pulmonary circulation of the anesthetized dog. The drugs were studied in the absence and presence of pro-pranolol (1 mg/kg, i.v.) in order to assess β-adrenoceptor-mediated effects in the pulmonary circulation. Intra-arterial administration of dopamine, (±)-dobutamine and (–)-dobutamine elicited dose-dependent increases in pulmonary perfusion pressure, reflecting increases in pulmonary vascular resistance. In control animals, dopamine elicited the largest increases in pulmonary perfusion pressure (45% above resting pulmonary pressure) followed by (–)-dobutamine (30% increase) and (±)-dobutamine (15% increase). The pressor effects of dopamine in the pulmonary circulation were mediated by both postjunctional vascular α1-and α2-adrenoceptors, since prazosin, (100 μg/kg, i.v.), a selective α1-adrenoceptor antagonist, and rauwolscine (100 μg/kg, i.v.), a selective α1-adrenoceptor antagonist, both inhibited the vasopressor response elicited by dopamine to roughly equivalent degrees. Pulmonary vasoconstriction produced by (±)-dobutamine and (–)-dobutamine was mediated primarily by postsynaptic vascular α1-adrenoceptors, although α2-adrenoceptor-mediated vasoconstriction was observed. In propranolol-pretreated animals, the increase in pulmonary perfusion pressure elicited by dopamine and (–)-dobutamine was qualitatively and quantitatively similar to that observed in control animals, suggesting that these agents have little activity on vascular β2-adrenoceptors in the pulmonary circulation. In marked contrast, the maximum pulmonary vasopressor response obtained with (±)-dobutamine were greater in propranolol-pretreated animals, indicating that (±)-dobutamine also has the capacity to stimulate pulmonary vascular β2-adrenoceptors which mediate pulmonary vasodilation that, in part, mask α-adrenoceptor-mediated pulmonary vasoconstriction. Since the (–)-enantiomer of dobutamine has little or no β2-adrenoceptor agonist activity, the β2-adrenoceptor-mediated effect of (±)-dobutamine must result from the (+)-enantiomer as has been previously proposed. The results of the present study indicate that dopamine has a greater propensity for increasing pulmonary vascular resistance, and therefore pulmonary arterial blood pressure, relative to (±)-dobutamine. This results, at least in part, from the relatively weaker activity of dopamine in stimulating pulmonary vascular β2-adrenoceptors which mediate vasodilation. This, in turn, shifts the balance of activity for dopamine in favor of activating pulmonary vascular α1 and α2-adrenoceptors to produce vasoconstriction. The results are consistent with, and may even account for, the clinical observation that dopamine is far more likely to increase pulmonary capillary wedge pressure and pulmonary vascular resistance in patients with congestive heart failure than is (±)-dobutamine, and that the β2-adrenoceptor agonist activity of (±)-dobutamine may be critical for the beneficial effects of this drug in the pulmonary circulation.