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We have developed an assay for studying myocardial phospholipase A2activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This assay was used to characterize phospholipase A2activity in rabbit myocardium. Lyophilized rabbit myocardium was incubated at 37°C in Tris-HCl buffer containing either ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl-lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-lysoplasmenylcholine were measured using a recently developed HPLC method. The identity of the individual species was confirmed by ion-spray LC-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min caused a linear increase in all lysophospholipids. The main increases were found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 ± 3 (mean ± SE, N= 8) and 48 ± 3 nmol/g dry wt × min, respectively. The apparent phospholipase A2activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0, and incubation at room temperature and 45°C decreased the activity by 80 and 40%, respectively. Studies of the metabolism of the formed lysophospholipids showed a substantial metabolism of the lysophospholipids that accounted for about 40% of the total phospholipase A2activity. This method offers a novel approach to study phospholipase A2activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools.

Abstract. The in vivo synthesis of thromboxane A2and prostacyclin was estimated in 23 diabetics through measurements of the major urinary metabolites 2,3-dinor-thromboxane B2and 2,3-dinor-6-keto-PGF1αutilizing gas chromatography-mass spectrometry. Mean excretion was similar to that in non-diabetic subjects. The possible influence of hyperglycemia on the excretion of 2,3-dinor-thromboxane B2and 2,3-dinor-6-keto-PGF1αwas evaluated in three ways: by measuring excretion before and during an acute 9-h normalization of hyperglycemia through an artificial pancreas (Biostator) as well as by comparing excretion before and 7–12 days or 40–180 days after the initiation of insulin treatment. Despite significant reducing effects on hyperglycemia or on levels of hemoglobin A1c, no effects on the excretion of the thromboxane and prostacyclin metabolites could be found. Abnormal formation of thromboxane or prostacyclin is not a generalized feature of the diabetic state.

Phospholipase A2 (PLA2) activity results in the formation of lysophospholipids and free fatty acids which may contribute to ischemic myocardial dysfunction. We evaluated the cardioprotective activity of two putative PLA2 inhibitors, quinacrine and 7,7-dimethyleicosadienoic acid (DEDA), in isolated globally ischemic rat hearts. Pretreatment with 1, 5 and 50 microM quinacrine before ischemia did not alter coronary flow but did cause significant cardiodepression. Twenty five minutes of global ischemia and 30 min of reperfusion caused severe myocardial dysfunction and lactate dehydrogenase release. Quinacrine significantly improved reperfusion contractile function and reduced lactate dehydrogenase release, indicative of cardioprotection. In contrast, 30 to 100 microM DEDA produced neither preischemic cardiodepression nor cardioprotective activity. PLA2 inhibition was inferred from measurements of the prostacyclin metabolite, 6-keto-prostaglandin F1 alpha in the coronary effluent and myocardial palmitoyl-lysophosphatidylcholine. Quinacrine and DEDA reduced both 6-keto-prostaglandin F1 alpha and palmitoyl-lysophosphatidylcholine by similar degrees. These results suggest that the cardioprotective activity of quinacrine is independent of PLA2 inhibition. A possible role of calcium inhibition was investigated in rat aortic smooth muscle strips. Norepinephrine-, KCl- and BAY K8644-induced contractions were antagonized in the presence of 5 and 50 microM quinacrine, but were unaffected by 30 to 60 microM DEDA. The ability of quinacrine to inhibit calcium was investigated further in cardiac ventricular myocytes. Measurement of mean whole cell calcium currents showed that quinacrine (5 microM) could inhibit this current up to 70%. Thus, these results suggest that quinacrine-induced cardioprotection may not be due to PLA2 inhibition, but may be related to calcium entry blocking activity.

The effects of intravenous infusions of the stable prostaglandin analogue 9-deoxo-16,16-dimethyl-9-methylene-PGE2 (9-methylene-PGE2) in a dosage of 10 or 24 µg/min were studied in the consicious euhydrated, dehydrated, and hyperhydrated with the simultaneous administration of exogenous arginine vasopressin (AVP), sheep. The infusions decreased urine osmolality and increased urine flow and renal free water clearance. The results indicate that 9-methylene-PGE2 exhibits its diuretic effect by antagonizing the antidiuretic action of AVP. In the hyperhydrated sheep receiving AVP the syndrome of inappropriate antidiuretic hormone release (SIADH) was simulated. As the prostaglandin analogue effectively blocked the antidiuretic effect of the AVP-administration it appears that 9-methylene-PGE2 may play a future role as a diuretic agent, especially in conditions characterized by water retention and dilutional hyponatremia such as SIADH.