Mark Rosolowsky

Local platelet activation causes vasoconstriction of large epicardial canine coronary arteries in vivo. Thromboxane A2 and serotonin are possible mediators.

The goal of the present study was to demonstrate that intracoronary platelet deposition may trigger intense vasoconstriction of large epicardial coronary arteries in vivo and that this is largely mediated by thromboxane A2 and serotonin released by activated platelets. Cyclic flow variations (progressive declines in blood flow followed by sudden restorations of flow) due to recurrent intracoronary platelet activation and thrombus formation were induced by damaging the endothelium and placing a cylindrical constrictor on the left anterior descending coronary artery (LAD) in open-chest, anesthetized dogs. Coronary diameters were measured in vivo by means of ultrasonic crystals sutured on the LAD immediately distal to the constrictor (LAD1) and 1 cm below (LAD2) and on the circumflex coronary artery (Cx). Coronary artery diastolic diameters were measured continuously before and during cyclic flow variations and after they were abolished by administration of LY53857, a serotonin-receptor antagonist (group 1, n = 7), or SQ29548, a thromboxane-receptor antagonist (group 2, n = 7). During cyclic flow variations, at the nadir of coronary flow, LAD1 (a site of maximal platelet accumulation) cross-sectional area decreased by 52 +/- 10% and 38 +/- 6% in group 1 and 2 animals, respectively (p less than 0.001 compared with values recorded during a brief LAD occlusion obtained by a suture snare), whereas LAD2 (a site of minimal or no platelet accumulation) cross-sectional area did not differ from that recorded during the brief LAD occlusion. SQ29548 abolished cyclic flow variations in seven of seven dogs and LY53857 in six of seven, but they affected the increased coronary vasoconstriction differently: LAD1 cross-sectional area increased by 32 +/- 6% of the control value in SQ29548-treated animals, whereas it returned to baseline dimension values in the LY53857-treated group as these interventions also abolished the cyclic flow variations. We conclude that a marked coronary vasoconstriction may be triggered by local platelet deposition and that thromboxane A2 and serotonin are mediators of this vasoconstriction.

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [14C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F1 alpha, PGE2, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11-hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF1 alpha and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF1 alpha exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.

Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries.

Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.

ADP plays an important role in mediating platelet aggregation and cyclic flow variations in vivo in stenosed and endothelium-injured canine coronary arteries.

The goal of this study was to test the hypotheses that endogenous ADP plays an important role in vivo in mediating platelet aggregation and cyclic coronary artery blood flow variations (CFVs) in stenosed and endothelium-injured coronary arteries in an experimental canine model. Anesthetized animals were studied and coronary blood flow velocities monitored by a pulsed Doppler flow probe positioned around the left anterior descending coronary artery. CFVs were established by an external constrictor positioned at sites with injured endothelium. Apyrase, an ADP-removing enzyme, was infused into the left anterior descending coronary artery (0.3-1.8 units/min) 30 minutes or 2 hours after the establishment of CFVs. Complete abolition of CFVs was achieved in 81% (13/16) of dogs with 30-minute CFVs and in 83% (five of six) of dogs with 2-hour CFVs. In other dogs, a potent inhibitor of ADP-induced platelet aggregation, clopidogrel, was administered as a 10 mg/kg i.v. bolus and a 2.5 mg/kg/hr infusion 30 minutes and 3 hours after the establishment of CFVs. This treatment resulted in complete abolition of CFVs in 14 dogs (100%) with either 30-minute or 3-hour CFVs. Epinephrine was infused into some dogs after CFVs had ceased as a result of either apyrase or clopidogrel administration and into some dogs in whom SQ29548, a thromboxane A2 receptor antagonist, had been given when apyrase failed to abolish CFVs. Epinephrine restored CFVs in all dogs treated with apyrase alone, 67% (four of six) of dogs treated with the combination of apyrase and SQ29548, and 29% (two of seven) of dogs treated with clopidogrel. The plasma epinephrine levels required for CFV restoration were 20 times higher than baseline values in dogs receiving apyrase alone, 100 times higher when a combination of apyrase and SQ29548 had been given, and more than 5,000 times higher in dogs receiving clopidogrel. In vitro studies showed that apyrase only inhibited ADP-induced platelet aggregation, whereas clopidogrel not only inhibited ADP-induced platelet aggregation, but also reduced platelet aggregation induced by the thromboxane mimetic U46619 and serotonin. These data suggest that 1) ADP is an important mediator of platelet aggregation and CFVs in vivo and 2) combined inhibition of thromboxane A2 and ADPs effects provides marked protection against CFVs in experimentally stenosed and endothelium-injured canine coronary arteries. These data and our previous observations are consistent with the possibility that specific antagonists of thromboxane A2, serotonin, and ADP, alone and together, may provide substantial protection against platelet aggregation leading to CFVs at sites of endothelial injury and coronary artery stenosis.

Products of heme-catalyzed transformation of the arachidonate derivative 12-HPETE open S-type K+ channels in Aplysia

In Aplysia mechanosensory neurons, the neuropeptide FMRFamide increases the opening of the background S-K+ channel. This action is mediated by activation of arachidonic acid metabolism. Arachidonic acid in Aplysia nervous tissue is transformed through the 12-lipoxygenase pathway to 12-HPETE, which undergoes further metabolism. In intact sensory cells, 12-HPETE simulates the FMRFamide response, raising the question of whether 12-HPETE is the messenger molecule ultimately acting on the S-K+ channel. Here we show that in cell-free (inside-out) patches from sensory cells, 12-HPETE fails to modulate the S-K+ channel, but in the presence of hematin (which catalyzes 12-HPETE metabolism), it triggers sharp increases in the channel opening probability. We also found that SKF-525A, an inhibitor of the cytochrome P450, reduces the response to FMRFamide, arachidonic acid, and 12-HPETE in intact cells. We conclude that a heme-catalyzed transformation of 12-HPETE is necessary and sufficient to promote the opening of the S-K+ channel and a heme-containing enzyme such as cytochrome P450 might play this key role.

Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function

During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer. During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated. Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs. Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence. The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs. When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence. However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence. Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs. We examined the effects of the EETs on PMN aggregation. 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal. 5,6-, 11,2- and 14,15-EETs also stimulated aggregation. FMLP stimulated the production of superoxide; however, 8,9-EET did not. These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation. Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.

Metabolism of arachidonic acid by canine polymorphonuclear leukocytes synthesis of lipoxygenase and omega-oxidized metabolites

Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B4 (LTB4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B2 (TXB2), 5-HETE, and several isomers of LTB4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12(S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12(R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.

Combined thromboxane A2synthetase inhibition and receptor blockade are effective in preventing spontaneous and epinephrine-induced canine coronary cyclic flow variations☆

The purpose of this study was to test the hypothesis that combined thromboxane A2 synthetase inhibition and receptor blockade is superior to either action alone in preventing cyclic flow variations in stenosed and endothelially injured canine coronary arteries. Forty-five dogs developed coronary cyclic flow variations after a plastic constrictor was placed around the left anterior descending coronary artery at the site where the endothelium was injured and received different interventions. In Group I, 17 dogs were treated with SQ 29,548, a thromboxane A2-prostaglandin H2 receptor antagonist. In Group II, 11 dogs received dazoxiben, a thromboxane A2 synthetase inhibitor. In Group III, R 68,070, a dual thromboxane A2 synthetase inhibitor and thromboxane A2-prostaglandin H2 receptor antagonist, was administered to 11 dogs. Group IV comprised six dogs that received aspirin before receiving R 68,070. Complete abolition of cyclic flow variations was achieved in 71% of dogs in Group I, 82% in Group II, 100% in Group III (p = 0.06 compared with Group I) and 50% in Group IV (p = 0.03 compared with Group III). Epinephrine was infused into dogs with abolished cyclic flow variations: all dogs in Group I had cyclic flow variations restored, 44% in Group II (p = 0.01 compared with Group I) and 64% in Group III (p = 0.04 compared with Group I). The plasma epinephrine levels required to restore cyclic flow variations were 2.2 +/- 0.5 ng/ml (control 0.04 +/- 0.01) in Group I, 8.7 +/- 4.5 ng/ml (control 0.05 +/- 0.02) in Group II and 7.4 +/- 2.6 ng/ml (control 0.07 +/- 0.02) in Group III.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1 alpha levels were significantly increased with respect to control and SQ 29548-treated dogs. Thus, simultaneous inhibition of TxA2 synthase and blockade of TxA2/PGH2 receptors is more effective than either intervention alone in this experimental model in enhancing thrombolysis and preventing reocclusion after t-PA administration.

Mediators of Arachidonic Acid–Induced Relaxation of Bovine Coronary Artery

Bovine coronary arteries relax in response to bradykinin, methacholine, sodium nitroprusside, isoproterenol, and arachidonic acid in a concentration-dependent manner. The relaxations to methacholine, bradykinin, and arachidonic acid are lost when endothelium is removed. Indomethacin, a cyclooxygenase inhibitor, attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus indomethacin) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus 2 x 10(-9) mo1/L, and 3 x 10(-7) versus 2 x 10(-6) mo1/L, respectively). Nitro-L-arginine, a nitric oxide synthase inhibitor, also attenuated the relaxations to methacholine, bradykinin, and arachidonic acid and shifted the EC50 (control versus nitro-L-arginine) to each (1 x 10(-7) versus 3 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). The combination of indomethacin and nitro-L-arginine blunted the relaxations to these agents and also shifted the EC50 values (control versus indomethacin plus nitro-L-arginine) to each (1 x 10(-7) versus 5 x 10(-7) mo1/L, 3 x 10(-10) versus > 10(-9) mo1/L, and 3 x 10(-7) versus > 10(-6) mo1/L, respectively). Methacholine, bradykinin, and arachidonic acid stimulated the release of prostaglandin I2, measured as 6-keto-PGF1 alpha. Indomethacin, but not nitro-L-arginine, inhibited arachidonic acid-induced release of 6-keto-PGF1 alpha. Vascular cGMP content was unchanged by arachidonic acid but was significantly elevated by bradykinin. Relaxations to prostaglandin I2 and sodium nitroprusside, but not 8,9-epoxyeicosatrienoic acid or isoproterenol, were inhibited by nitro-L-arginine. We conclude that the endothelium-dependent relaxations to methacholine, bradykinin, and arachidonic acid are partly due to prostaglandin I2 release. The remainder of the responses to these agents is due to the release of other relaxing factor or factors. Since bradykinin increased cGMP and nitro-L-arginine partially inhibited its relaxant effects, nitric oxide also appears to participate in the bradykinin-induced effect. Since the combination of indomethacin and nitro-L-arginine failed to completely block the relaxations to methacholine, bradykinin, and arachidonic acid, another endothelial factor must contribute to their vascular effects. Surprisingly, nitro-L-arginine attenuated the relaxations to arachidonic acid; however, L-arginine failed to reverse the effects of nitro-L-arginine on arachidonic acid-induced relaxations. In addition, arachidonic acid failed to increase cGMP. Nitro-L-arginine also reduced the responses to prostaglandin I2 and sodium nitroprusside. These data indicate that these arginine analogues may have effects other than competitive inhibition of nitric oxide synthase.