David M. Roth

Studies on the mechanism of leukotriene induced coronary artery constriction

Leukotriene (LT) C4, D4, and E4 at concentrations of 10 to 100 ng/ml were found to be potent coronary artery constrictors in the perfused cat coronary artery and perfused rat heart. In contrast, LTB4, was essentially inactive. The coronary constrictor effect of leukotrienes was not related to thromboxane release, but rather appeared to be due to a calcium mediated activation of specific leukotriene receptors.

Vascular responsiveness and eicosanoid production in diabetic rats

SummaryVascular responsiveness to vasoactive eicosanoids as well as vascular prostacyclin and thromboxane production was investigated in 7–10 weeks alloxan-diabetic rats. Aortic rings from diabetic rats exhibited increased responsiveness to carbocyclic thromboxane A2, a thromboxane analogue, when compared to control rat aortae. Isolated perfused hearts of diabetic rats showed increased vascular responsiveness to 9,11-methanoepoxy PGH2 (U-46619), an endoperoxide analogue. Diabetes resulted in a reduction in prostacyclin generation by isolated incubated aortae which was overcome by the addition of arachidonic acid but not by homogenization of incubated aortic tissue. In contrast, prostacyclin, but not thromboxane, generation was elevated in isolated perfused hearts of diabetic animals in response to moderate doses of arachidonic acid, but at high doses of arachidonate, more thromboxane was formed by perfused hearts of diabetic rats. These results suggest that different vessels can either increase or decrease their prostaglandin production in response to diabetes. The alterations in prostanoid production may be due to differential changes in prostacyclin and thromboxane synthesis in vessels which, in turn, may be related to the changes in vascular responsiveness.

Potentiation of leukotriene formation in pulmonary and vascular tissue

SummaryLeukotriene (LT) release from vascular and pulmonary tissue was assessed by a radioimmunoassay for peptide leukotrienes (i.e., LTC4, LTD4 and LTE4). The calcium ionophore A-23187 at 1–3 μg/ml and platelet activating factor (PAF) at 10 μg/ml produced marked formation of peptide leukotrienes in minced cat pulmonary tissue. This was also confirmed by bioassay of the incubates in isolated perfused cat coronary arteries. Rat pulmonary tissue was comparable to cat with regard to LT production, but guineapig lung produced about 30–50% less on a weight basis. In addition, aortic and coronary artery vessel walls produced significant amounts of LTs. The time course for maximal leukotriene production occurred at 45–60 min of incubation at 37°C in both the radioimmunoassay and the bioassay. Cat coronary artery constricted markedly to LTC4 or LTD4 (30–40 mm Hg) and to the lung or blood vessel incubate. This constriction was virtually totally blocked by the leukotriene antagonist FPL-55712, but not by the thromboxane receptor antagonist, pinane thromboxane A2, the α-adrenergic receptor antagonist, phenoxybenzamine, or the angiotensin receptor antagonist, saralasin. Thus, pulmonary and vascular tissue produce leukotrienes that appear to exert coronary constrictor effects on specific leukotriene receptors. These results indicate that the ischemia of shock and anaphylaxis may be accentuated by the release of peptide leukotrienes.

Antagonism of platelet aggregation by 13-azaprostanoic acid in acute myocardial ischemia and sudden death

The effects of 13-azaprostanoic acid (13-APA) were studied during acute myocardial ischemia in cats and in rabbit sudden death induced by sodium arachidonate (Na-Ar). To more clearly define the mechanism of action of 13-APA, we also examined its effects on isolated cat and rabbit coronary arteries, in vitro aggregation of cat and rabbit platelet-rich plasma (PRP) and circulating rabbit platelet count measured in vivo. 13-APA provided minimal protection during myocardial ischemia in cats, partially reversing ischemia-induced ST segment elevations by 3-5 hours after coronary artery occlusion. However, 13-APA was ineffective in inhibiting the rise in plasma creatine kinase (CK) activity or the loss of CK from ischemic myocardial tissue. 13-APA (1.0 - 100 microM) did not inhibit contraction of cat coronary arteries produced by a stable thromboxane A2 analog. However, 13-APA (100 microM) inhibited aggregation of cat PRP induced by AA (1.0 microM). 13-APA also provided significant protection against sudden death induced by Na-Ar in rabbits. While this agent was ineffective in reducing vasoconstriction of rabbit coronary arteries or inhibiting platelet aggregation in response to 500 microM AA, aggregation of rabbit PRP by 250 microM AA was completely inhibited. AA injection produced a significant decrease in circulating platelet count in vehicle-treated rabbits. However, 13-APA reduced the decrease in circulating platelet count in rabbits which survived AA injection during the 13-APA infusion. These results indicate that antagonism of thromboxane A2 receptors in platelets may be an important feature in protecting against sudden death. The difference in sensitivities of vascular and platelet thromboxane receptors as well as the accessability of 13-APA to these receptors may explain the lack of protection of 13-APA in myocardial ischemia.

Leukotriene production in isolated tissues of diabetic rats

We examined the ability of heart, aorta and lung obtained from alloxan diabetic rats as well as control rats to produce peptide leukotrienes (LT). The isolated perfused heart preparation as well as incubated minced tissue preparations were studied. Upon infusion of the Ca++ ionophore A23187, hearts from diabetic rats produced significantly less peptide LT when compared to control hearts. Lung tissue from diabetic animals incubated with A23187 also produced less immunoreactive peptide leukotrienes (iLT) when compared to the control group. In both preparations, incubation with the lipoxygenase inhibitor propyl gallate significantly inhibited the production of iLT in both the diabetic and control group. The observed differences in production of leukotrienes may alter vascular reactivity and thus play a role in the cardiovascular complications observed in diabetes.

Modulation of leukotriene synthesis and actions by synthetic derivatives of arachidonic acid.

Seven analogs of arachidonic acid were tested for their coronary vasoactivity and their ability to inhibit LTC4 and LTD4 synthesis by lung tissue and to antagonize LTD4 induced coronary constriction. None of the seven arachidonic acid analogs significantly altered peptide leukotriene production by minced cat lung. Two of the analogs (i.e., 7, 13-diethanoarachidonic acid and 7, 10, 13-triethanoarachidonic acid) exerted modest but significant coronary vasodilation in isolated cat coronary arteries, and significantly antagonized the coronary vasoconstrictor response to LTD4. These analogs may be of interest in modulating leukotriene actions.

Specificity of anti-leukotriene actions of nicardipine

The calcium channel blocker, nicardipine (100 ng/ml) markedly antagonized the coronary vasoconstrictor effect of the peptide leukotrienes LTC4 and LTD4 on the isolated perfused cat coronary artery. However, nicardipine even at 300 ng/ml failed to antagonize the leukotriene induced contraction of either tracheal or pulmonary parenchymal strips from guinea pigs. However, at higher concentrations (i.e., 10 micrograms/ml), nicardipine inhibited the production of peptide leukotrienes from minced cat lung incubated in the presence of A23187. Thus, nicardipine exerts some selectivity in its anti-leukotriene actions.

Protective Actions of Ibuprofen in Arachidonate-Induced Sudden Death

Sodium arachidonate given intravenously at a dose of 2 mg/kg is uniformly lethal in rabbits. Rabbits die within 2-5 min following a dramatic decrease in mean arterial blood pressure (MABP), and in circulating platelets, and a large increase in plasma thromboxane B2 (TxB2) concentration. The nonsteroidal anti-inflammatory agent, ibuprofen, given 15 min prior to challenge with sodium arachidonate, significantly protects against the abrupt decrease in MABP and in circulating platelets and prevents the increase in circulating TxB2 concentrations. These rabbits all survive the lethal effects of arachidonic acid when given ibuprofen at doses of 0.75, 6.25 or 12.5 mg/kg intravenously 15 min prior to arachidonate challenge. At 0.375 mg/kg, only 20% of the animals survive. Concomitant with survival is a significant attenuation of the decrease in MABP (84 +/- 8 mm Hg without ibuprofen vs. 1 +/- 1 to 33 +/- 12 mm Hg with 12.5-0.75 mg/kg ibuprofen). Similarly, these rabbits show a greatly reduced loss of circulating platelets, and virtually no increases in the formation of TxB2. Ibuprofen protects against arachidonate-induced sudden death in a dose-related manner. The mechanism of the protection appears to involve prevention of adherence or aggregation of platelets and the subsequent formation of thromboxane A2. The net result is prevention of pulmonary thrombosis, the major pathological event in triggering sudden death.

Modulation of receptor mediated leukotriene release in the perfused heart

The chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) induces peptide leukotriene release at concentrations of 20-25 pmol/ml 3 min after the start of FMLP infusion. FMLP-induced leukotriene release in rabbit hearts is not blocked by the leukotriene receptor antagonist FPL-55712 at concentrations that totally antagonize the hemodynamic effects of exogenously infused peptide leukotrienes. Moreover, propyl gallate, a lipoxygenase inhibitor, does not block FMLP-induced leukotriene release. However, the chemotactic peptide antagonist (Boc-Phe-Leu-Phe-Leu-Phe-OH) totally antagonized FMLP-induced leukotriene release suggesting that the release is via a different mechanism, possibly a receptor mediated event.

Cardiac Effects of Peptidoleukotrienes

The leukotrienes are a group of newly identified eicosanoids of the lipoxygenase pathway of arachidonic acid metabolism (Samuelsson, 1981). The peptide-con-taining leukotrienes (LT) (i. e., LTC4, LTD4, and LTE4) are known to possess potent biological properties including bronchoconstriction (Holroyde et al., 1981), enhancement of vascular permeability (Peck et al., 1981), and vasoconstriction (Yokochi et al., 1982). In this regard, it is well documented that leukotrienes C4and D4 are potent coronary constrictors in a number of animal species (Woodman and Dusting, 1983; Terashita et al., 1981). Previous work in our laboratory has shown that the leukotrienes are potent constrictors of isolated perfused cat coronary arteries (Roth and Lefer, 1984). Leukotrienes have also been claimed to exert direct negative inotropic effects on the myocardium (Burke et al., 1982). The purpose of this investigation was to examine the inotropic effects of the leukotrienes in relation to their potent coronary constricting activity and to clarify the relationship between coronary vasoactivity and inotropicity in cardiac preparations isolated from three commonly used mammalian species.