William M. Mackin

Effect of azapropazone and allopurinol on myocardial infarct size in rats

The effect of the xanthine oxidase inhibitor allopurinol and the non-steroidal antiinflammatory agent azapropazone on infarct size in rats, subjected to 48 h of occlusion of the left anterior descending coronary artery, were studied. Allopurinol (50 mg/kg i.p., twice daily from 24 h before to 48 h after LAD occlusion) and azapropazone (100 mg/kg i.p twice daily from 24 h before to 48 h after LAD occlusion) significantly reduced infarct size when compared to saline-treated rats. These data point towards involvement of xanthine oxidase derived free radicals in evolving myocardial infarction in rats; beneficial effect of azapropazone in this model may be related to the drugs ability to inhibit xanthine oxidase as well as various key neutrophil functions.

Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug

Azapropazone at concentrations of 0.1 to 1 mM inhibited by 30-70% rat neutrophil migration, aggregation, and degranulation in response to the synthetic chemotactic peptide fMet-Leu-Phe. Binding studies using fNle-Leu-[3H]Phe, a radiolabeled analog of fMet-Leu-Phe, showed that azapropazone did not inhibit these responses by interfering with fMet-Leu-Phe binding. Azapropazone also decreased both the apparent rate of production and maximal levels of superoxide anion (O2-) generated by cells stimulated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA). The concentrations of azapropazone that inhibit these neutrophil responses in vitro approximate those previously found in vivo after administration of therapeutic doses of drug to rats or humans. Taken together, the data suggest that the efficacy of azapropazone in gouty arthritis may be partly due to its ability to inhibit key neutrophil functional responses in vivo.

Acute lung inflammation in rats induced by phorbol myristate acetate (PMA)

Intratracheal administration of PMA produces acute lung injury in part due to the generation of O2-derived free radicals. This study evaluated the role of the antioxidant enzyme superoxide dismutase (SOD) in PMA-induced lung injury in the rat. PMA was instilled into rats intratracheally (20–60 μg/kg), and the lungs were lavaged 4 hr later. Total number of cells recovered from lavage after PMA treatment was not different from the total number recovered from controls; lavagable PMNs increased in a dose-dependent manner. Albumin in lavage fluid (an index of lung vascular permeability) was significantly increased at 60 μg/kg PMA. SOD (10,000 U)+PMA (60 μg/kg) reduced the albumin level but significantly increased both total number of cells and number of PMNs recovered from lavage fluid. To investigate the possibility that SOD decreases the ability of PMNs to adhere, PMN aggregation was measuredin vitro. The results indicated that 10,000 U SOD can inhibit PMA-induced aggregation by 50%. In contrast, aggregation to other stimuli (e.g., fMet-Leu-Phe, A23187) was unaffected by SOD. We conclude SOD prevents PMA-induced lung permeability and diminishes PMN adherence.

Cellular and Biochemical Characterization of the Anti-Inflammatory Effects of DuP 654 in the Arachidonic Acid Murine Skin Inflammation Model (Part 1 of 2)

The possible utility of DuP 654, a potent 5-lipoxygenase inhibitor, for treating human inflammatory skin disease was investigated in murine skin treated with 1.0 mg arachidonic acid (AA). When DuP 654 was applied to murine skin treated with AA, it inhibited the resulting inflammation and influx of cells. High performance liquid chromatography and radioimmunoassay analysis of lipid extracts from AA-treated ears indicated that the influx of polymorphonuclear leukocytes (PMN) was temporally preceded by an appearance of significant amounts of 5-HETE (6.7 +/- 1.4 ng/ear) and Leukotriene B4 LTB4 0.92 +/- 0.2 ng/ear) when compared with extracts of untreated ears (5-HETE, 02 +/- 0.3 ng/ear; LTB4, less than 0.1 ng/ear). The levels of the 5-lipoxygenase products were reduced by treatment with 10 micrograms/ear DUP 654. Lipid extracts from AA-treated ears contain chemotactic activity for human PMN and this chemotactic activity in the AA-treated ears could be reduced but not eliminated by immunosorption with anti-LTB4 antibodies coupled to protein A-agarose. The appearance of the chemotactic activity was inhibited by DuP 654. Taken together, these data suggest that DuP 654 may have utility in human inflammatory skin disease.

Biochemical and Pharmacologic Characterization of a Phosphatidylinositol-Specific Phospholipase C in Rat Neutrophils

The phosphatidylinositol (PI)‐specific phospholipase C (PLC) activity contained in sonicates of casein‐elicited (4‐6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15‐20 min at 37° and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5‐6.0, is enhanced 1.5‐3‐fold by the addition of deoxycholate, and is Ca+ + dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 ± 85 μM and a Vmax of 34.3 ± 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 ± 66 μM and a Vmax of 14.3 ± 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values 100 μM) inhibitors of the PMN PLC.

Persistent cardioprotection by azapropazone in a canine model of coronary artery thrombosis and thrombolysis

Activated neutrophils and possibly xanthine oxidase-derived free radicals are believed to he mediators of ischemia and reperfusion-induced myocardial damage. We studied the cardioprotective effect of the neutrophil stabilizer and xanthine oxidise inhibitor azapropazone in dogs subjected to thrombotic occlusion of the left anterior descending coronary artery (LAD), induced by intracoronary introduction of a copper coil, followed 60 min later by thrombolytic treatment with intracoronary streptokinase and 4-day reperfusion; we then determined infarct size by triphenyltetrazolium stain. Azapropazone [100 mg/kg intravenously (i.v.) followed by a 24-h i.v. infusion of 10 mg/kg/h, n = 8] or vehicle (n = 10) treatments were started immediately before the streptokinase infusion. Steady-state plasma levels of azapropazone ranged from 97 to 163 μg/ml during the infusion. Myocardial blood flow and underperfused area at risk were determined using radiolaheled microspheres. Results were as follows (mean ± SEM): area at risk (percentage of left ventricle) azapropazone 22.7 ± 3.16 and vehicle 21.8 ± 4.13; infarct size (percentage of area at risk), azapropazone 45.1 ± 11.8 and vehicle 75.7 ± 10.6, p < 0.03; collateral blood flow (ml/min/g), azapropazone 0.27 ± 0.02 and vehicle 0.23 ± 0.02: total ischemic period (min), azapropazone 106 ± 5.9 and vehicle 91.5 ± 4.9. Azapropazone had no effects on heart rate (HR), blood pressure (BP), or rate/pressure product (RPP). These data show that azapropazone limits infarct size in a canine model of coronary thrombosis and long-term reperfusion and that this cardioprotection is independent of cardiovascular parameters.

Effects of GTPγS and fMet-Leu-Phe on rat PMN phospholipase C (PLC) activity

The PLC activities associated with the cytosol and plasma membrane subcellular fractions of rat PMN were tested for sensitivity to stimulation by GTPγS and/or fMet-Leu-Phe. PMN plasma membrane PLC was stimulated 10–20% by 50 μM GTPγS (p<0.02). fMet-Leu-Phe alone had no effect on the plasma membrane PLC activity, but addition of fMet-Leu-Phe and GTPγS together stimulated PLC activity by 20–30% (p<0.05). Neither GTPγS nor fMet-Leu-Phe had any significant effect on the cytosolic PLC activity. These data demonstrate the presence of a PLC activity in rat PMN plasma membranes that is sensitive to stimulation by GTPγS and fMet-Leu-Phe.

THE PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C (PLC) IN RAT NEUTROPHILS (PMN)

ABSTRACT The phosphatidylinositol (Pl)-specific PLC contained in crude sonicates of casein elicited rat neutrophils has been identified and biochemically characterized.