(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2.

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2­(R)-naproxen and COX-2­(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein-coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2-/-). EP2-/- mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2-/- mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2-/- mice developed profound systolic hypertension, whereas wild-type mice showed no change in systolic blood pressure. Analysis of wild-type and EP2-/- mice on day 5 of pregnancy indicated that the reduced litter size of EP2-/- mice is due to a pre-implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt-sensitive hypertension, and also plays an essential part in female fertility.

Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A2.

When human platelets are aggregated by thrombin, material is released that rapidly contracts strips of spirally cut porcine coronary artery. Prevention of the contraction by indomethacin suggested mediation by a prostaglandin. The contraction produced by aggregating platelets was unlike those produced by prostaglandins E2, F2alpha, G2, or H2, but resembled that evoked by thromboxane A2, which is formed by platelets during aggregation.

Tolerance to the humoral and hemodynamic effects of caffeine in man.

Acute caffeine in subjects who do not normally ingest methylxanthines leads to increases in blood pressure, heart rate, plasma epinephrine, plasma norepinephrine, plasma renin activity, and urinary catecholamines. Using a double-blind design, the effects of chronic caffeine administration on these same variables were assessed. Near complete tolerance, in terms of both humoral and hemodynamic variables, developed over the first 1-4 d of caffeine. No long-term effects of caffeine on blood pressure, heart rate, plasma renin activity, plasma catecholamines, or urinary catecholamines could be demonstrated. Discontinuation of caffeine ingestion after 7 d of administration did not result in a detectable withdrawal phenomenon relating to any of the variables assessed.

Estimated rate of prostacyclin secretion into the circulation of normal man.

The rate of secretion of prostacyclin (PGI2) into the circulation of normal man was estimated by measurement of the 2,3-dinor-6-keto-PGF1 alpha (D) and 15-keto-13,14-dihydro-2,3-dinor-6-keto-PGF1 alpha (KDD) urinary metabolites of PGI2. Subjects received 6-h intravenous infusions of vehicle alone and PGI2 at 0.1, 0.4, and 2.0 ng/kg per min in random order. The fractional elimination of the metabolites was independent of the rate of PGI2 infusion. 6.8 +/- 0.3% of the infused PGI2 appeared as D and 4.1 +/- 0.4% as KDD. The regression of infused PGI2 upon the quantities of the two metabolites excreted in excess of control values permitted estimation of the rate of entry of endogenous PGI2 into the circulation corresponding to a given quantity of metabolite excreted. Using the quantities excreted in the 24 h from commencement of the infusions the estimated rates were 0.08 +/- 0.02 ng/kg per min from D and 0.10 +/- 0.03 from KDD. Studies with exogenous PGI2 suggest that infusion rates 2--4 ng/kg per min are required to achieve the threshold for inhibition of platelet function (ex vivo) in man. Although not precluding a role for PGI2 in local platelet-vessel wall interactions, the much lower estimates obtained in this study suggest that endogenous PGI2 is unlikely to act as a circulating antiplatelet agent in healthy man.

Guanethidine and related agents. 3. Antagonism by drugs which inhibit the norepinephrine pump in man.

Antagonism of the antihypertensive action of guanethidine by the tricyclic antidepressants, desipramine and protriptyline, has been demonstrated in controlled studies. These antidepressants also prevent the effect of the related ring-substituted guanidinium adrenergic neuron blockers, bethanidine and debrisoquin. That the rise in blood pressure when desipramine is added to guanethidine therapy is not due simply to a pressor action of the two drugs in combination was demonstrated by the lack of an increase in blood pressure when guanethidine was added to desipramine therapy. Investigations were conducted to determine whether antagonism of guanethidine's clinical effect could result from blockade by the tricyclic antidepressants of the norepinephrine pump in the adrenergic neuron membrane, thereby preventing the uptake of guanethidine into the neuron by this pump. Like guanethidine, the indirectly acting pressor amine, tyramine, enters the neuron via the norepinephrine pump. Desipramine, protriptyline, and amitriptyline in clinical doses all were found to block the pressor action of tyramine while potentiating the pressor effect of norepinephrine. The amino acid, methyldopa, does not enter the neuron via the norepinephrine pump, and its antihypertensive action is not altered by concomitant administration of tricyclic antidepressants. It is concluded from the evidence in this investigation together with the results of previous studies in experimental animals that clinical doses of desipramine-like drugs inhibit the norepinephrine pump in the peripheral adrenergic neuron in man and thereby prevent uptake of guanethidine to its site of action.

Effects of prostaglandin cyclic endoperoxides on the lung circulation of unanesthetized sheep.

Although prostaglandins E(2) and F(2alpha) have been suggested as mediators of the pulmonary hypertension seen after endotoxin infusion or during alveolar hypoxia, their precursors, the endoperoxides (prostaglandins G(2) and H(2)) are much more potent vasoconstrictors in vitro. In this study we compared the effects of prostaglandin (PG)H(2), a stable 9-methylene ether analogue of PGH(2) (PGH(2)-A), PGE(2), and PGF(2alpha) on pulmonary hemodynamics in awake sheep. The animals were prepared to allow for measurement of (a) lung lymph flow; (b) plasma and lymph protein concentration; (c) systemic and pulmonary vascular pressures; and (d) cardiac output. We also determined the effect of prolonged PGH(2)-A infusions on lung fluid balance and vascular permeability by indicator dilution methods, and by assessing the response of lung lymph. Both PGH(2) and PGH(2)-A caused a dose-related increase in pulmonary artery pressure: 0.25 mug/kg x min tripled pulmonary vascular resistance without substantially affecting systemic pressures. Both were 100 times more potent than PGE(2) or PGF(2alpha) in this preparation. PGH(2)-A, as our analysis of lung lymph and indicator dilution measurements show, does not increase the permeability of exchanging vessels in the lung to fluid and protein. It does, however, augment lung fluid transport by increasing hydrostatic pressure in the pulmonary circulation. We conclude: (a) that PGH(2) is likely to be an important mediator of pulmonary vasoconstriction; (b) its effects are probably not a result of its metabolites PGE(2) or PGF(2alpha).

Endogenous prostacyclin biosynthesis and platelet function during selective inhibition of thromboxane synthase in man.

The consequences of inhibiting the metabolism of prostaglandin G2 to thromboxane A2 in man were studied by using an inhibitor of thromboxane synthase, 4-[2-(IH-imidazol-1-yl)ethoxy] benzoic acid hydrochloride (dazoxiben). Single doses of 25, 50, 100, and 200 mg of dazoxiben were administered to healthy volunteers at 2-wk intervals in a randomized, placebo-controlled, double-blind manner. Serum thromboxane B2 and aggregation studies in whole blood and platelet-rich plasma were measured before dosing and at 1, 4, 6, 8, and 24 h after dosing. Both serum thromboxane B2 and the platelet aggregation response to arachidonic acid (1.33 mM) were reversibly inhibited in a dose-dependent manner. Aggregation induced by 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (0.4 and 4.0 microM) in platelet-rich plasma as well as both aggregation and nucleotide release induced by collagen (95 micrograms/ml) in platelet-rich plasma and whole blood were unaltered by dazoxiben. Additional evidence for a platelet-inhibitory effect of the compound was a significant prolongation of the bleeding time at 1 h after administration of the highest dose (200 mg) of dazoxiben. Endogenous prostacyclin biosynthesis was assessed by measurement of the major urinary metabolite of prostacyclin, 2,3-dinor-6-keto-PGF1 alpha (PGI-M). PGI-M excretion was increased by dazoxiben; it rose a mean 2.4-fold from predosing control values at 0-6 h after administration of the highest dose studied (200 mg).

Encainide and its metabolites. Comparative effects in man on ventricular arrhythmia and electrocardiographic intervals.

To assess the relative contributions of encainide and its putatively active metabolites, O-demethyl encainide (ODE) and 3 methoxy-O-demethyl encainide (3MODE), to the drug's pharmacologic effects, we compared intravenous infusions and sustained oral therapy in two phenotypically distinct groups of patients, extensive and poor metabolizers of encainide. Unlike poor metabolizers, extensive metabolizers had appreciable quantities of both metabolites detectable in plasma and had fourfold shorter elimination half-lives for encainide. By quantitating electrocardiogram intervals, arrhythmia frequency, and plasma concentrations, we found that, in poor metabolizers, arrhythmia suppression and ventricular complex (QRS) prolongation were correlated positively with encainide concentrations (r greater than or equal to 0.570, P less than 0.014). In these two subjects, antiarrhythmic concentrations of encainide (greater than 265 ng/ml) were at least fivefold higher than those sustained in the six extensive metabolizers during steady state oral therapy. In extensive metabolizers, encainide concentrations were uncorrelated with effects. Arrhythmia suppression and QRS prolongation in extensive metabolizers correlated best with ODE (r greater than or equal to 0.816, P less than 0.001); QTc change correlated positively with both 3MODE and ODE. Arrhythmia suppression paralleled QRS prolongation; the relationship between them appeared similar in both phenotypic groups. In most patients, extensive metabolizers, encainide effects during oral therapy are mediated by metabolites, probably ODE.

Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man.

To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.

Urinary prostaglandins. Identification and origin.

Human urine was analyzed by mass spectrometry for the presence of prostaglandins. Prostaglandin E2 and F2alpha were detected in urine from females by selected ion monitoring of the prostaglandin E2-methylester-methoxime bis-acetate and the prostaglandin F2alpha-methyl ester-Tris-trimethylsilylether derivative. Additional evidence for the presence of prostaglandin F2alpha was obtained by isolating from female urine an amount of this prostaglandin sufficient to yield a complete mass spectrum. The methods utilized permitted quantitative analysis. The origin of urinary prostaglandin was determined by stimulating renal prostaglandin synthesis by arachidonic acid or angiotensin infusion. Arachidonic acid, the precursor of prostaglandin E2, when infused into one renal artery of a dog led to a significant increase in the excretion rate of this prostaglandin. Similarly, infusion of angiotensin II amide led to a significantly increased ipsilateral excretion rate of prostaglandin E2 and F2a in spite of a simultaneous decrease in the creatinine clearance. In man, i.v. infusion of angiotensin also led to an increased urinary eliminiation of prostaglandin E. These results show that urinary prostaglandins may originate from the kidney, indicating that renally synthesized prostaglandins diffuse or are excreted into the tubule. Thus, urinary prostaglandins are a reflection of renal prostaglandin synthesis and have potential as a tool to delineate renal prostaglandin physiology and pathology.

Prostaglandin J2 and 15-deoxy-delta12,14-prostaglandin J2 induce proliferation of cyclooxygenase-depleted colorectal cancer cells.

Increased expression of cyclooxygenase (COX) and overproduction of prostaglandins (PGs) have been implicated in the development and progression of colorectal cancer (CRC). Nonsteroidal anti-inflammatory agents (NSAIDS) inhibit growth of various CRC cell lines by both COX-dependent and COX-independent pathways. To specifically examine the effect of COX and PGs on proliferation in CRC cells, we introduced an antisense COX-2 cDNA construct under the control of a tetracycline (Tc)-inducible promoter into a CRC cell line, HCA-7, Colony 29 (HCA-7) that expresses COX and produces PGs. In the presence of Tc, PG production in COX-depleted cells was reduced 99.8% compared with either uninduced transfectants or parental HCA-7 cells. This decrease in PG production was associated with a concomitant 60% reduction in DNA replication. Subsequently, we examined the effects of various PGs to modulate cell growth in COX-depleted HCA-7 or COX-null HCT-15 cells by quantifying [3H]thymidine incorporation and/or growth in collagen gels. We report that J-series cyclopentenone PGs, particularly PGJ2 and 15-deoxy-delta12,14-PGJ2, induce proliferation of these cells at nanomolar concentrations. Lipids extracted from parental HCA-7 cell conditioned medium stimulated mitogenesis in COX-depleted HCA-7 cells and COX-null HCT-15 cells. Using chromatographic and mass spectrometric approaches, we were able to detect PGJ2 in conditioned medium from parental HCA-7 cells. Taken together, these findings implicate a role for cyclopentenone PGs in CRC cell proliferation.

Oxygenation of arachidonic acid by hepatic microsomes of the rabbit. Mechanism of biosynthesis of two vicinal dihydroxyeicosatrienoic acids.

[1-14C] Arachidonic (eicosatetraenoic) acid was incubated at 37 degrees C for 15 min with rabbit liver microsomes fortified with NADPH (1 mM). The products were purified by high-pressure liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry. Based on polarity on reversed phase HPLC, the metabolites could be divided into three groups. The major metabolites of lowest polarity were 19- and 20-hydroxyarachidonic acid and 19-oxoarachidonic acid. The major metabolites of medium polarity were two diols, 14,15-dihydroxy-5,-8,11-eicosatrienoic acid and 11,12-dihydroxy-5,8,14-eicosatrienoic acid. Microsomal incubation under atmospheric isotopic oxygen led to incorporation of only one 18O molecule in each diol, indicating that the diols could originate from breakdown of 14(15)-oxido-5,8,11-eicosatrienoic acid and 11(12)-oxido-5,8,14-eicosatrienoic acid, respectively. Major metabolites in the most polar group were 14,15,19- and 14,15,20-trihydroxy-5,8,11-eicosatrienoic acid. 11,12,19- and 11,12,20-trihydroxy-5,8,14-eicosatrienoic acid and 11,12-dihydroxy-19-oxo-5,8,-14-eicosatrienonic acid. About 0.5% of exogenous radioactively labelled arachidonic was covalently bound to microsomal proteins. The metabolites and the protein-bound products were formed in considerably smaller amounts by non-fortified microsomes. Carbon monoxide inhibited this pathway of arachidonic acid metabolism, indicating that these reactions might be catalyzed by the cytochrome P-450-linked monooxygenase systems.

Enrichment of rat tissue lipids with fatty acids that are prostaglandin precursors.

The effects of supplementation of a complete diet with ethyl arachidonate and with ethyl dihomo-gamma-linolenate (20 : 3Omega6) on the fatty acid composition of plasma and tissue lipid classes were studied in normal rats. 2. These prostaglandin precursors were incorporated in varying degrees into all lipid classes of the tissues that were investigated. The largest elevations were seen in plasma and tissue triacylglycerols. Significant increases were also observed in phospholipids, cholesteryl esters and the free fatty acid fraction. 3. Following the feeding of the ester of 20 : 3Omega6, arachiodonate levels also rose in the lipids of some tissues. In others, such as the renal medulla and platelets, and increase in 20 : 3Omega6 content occurred without a rise in 20 : 4. 4. Platelet aggregation is known to be stimulated by 20 : 4 (via active metabolites), but not by 20 : 3Omega6. The ability to modify 20 : 3Omega6 levels selectively in certain tissues is of interest in light of such pharmacologic differences from 20 : 4.

Prostaglandin thromboxane, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells.

Production of several metabolites of arachidonic acid by purified rat serosal mast cells in response to stimulation with the ionophore A23187 was assessed by stable isotope dilution assay using gas chromatography-mass spectrometry. Compounds quantified were prostaglandins D2, E2, F2 alpha, 6-keto-F1 alpha, thromboxane B2, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Mast cells incubated at 37 degrees C for 30 min without ionophore produced measurable quantities of all metabolites assayed. 4 microM A23187 resulted in substantial increased synthesis of all metabolites compared to control cells. Of the metabolites quantified, prostaglandin D2 and prostacyclin were the major products derived from arachidonic acid in ionophore-stimulated rat mast cells.

Novel transformations of arachidonic acid by the rat thyroid in vitro.

The transformation of arachidonic acid by the rat thyroid in vitro has been investigated. At least two metabolites have been partially characterized: they differed from known metabolites of arachidonic acid in terms of retention volume in liquid chromatography, ultraviolet spectrophotometry and pharmacology (formation not inhibited by indomethacin and enhanced by eicosatetraynoic acid). The analysis by chemical ionization mass spectrometry suggested that these metabolites might be diketo-monohydroxy- and monoketo-dihydroxy-compounds. The conversion of arachidonic acid into these compounds was stimulated by ionophore A23187, decreased by the peroxidase inhibitor methimazole and potentiated by iodide, suggesting that this pathway is under the control of Ca2+ and of a peroxidase product.

Regional differences in prostacyclin formation by the kidney. Prostacyclin is a major prostaglandin of renal cortex.

Microsomes prepared from rabbit renal cortex were found to synthesize substantial amounts of 6-ketoprostaglandin F1alpha from prostaglandin G2 or arachidonic acid during an incubation. In contrast, no 6-ketoprostaglandin F1alpha was formed by renal medullary microsomes which synthesize predominantly prostaglandin E2. Mass spectral confirmation of the structure of 6-ketoprostaglandin F1alpha from these incubations demonstrates the ability of the renal cortex to synthesize prostacyclin.

Incorporation of 12(S)-hydroxyeicosatetraenoic acid into the phosphatidylcholine signaling pathway.

The incorporation of 12-lipoxygenase metabolites into phospholipids (PLs) could modify second messengers such as diacylglycerols (DAG) and phosphatidic acids. Incubation of [(14)C]12(S)-HETE (1 microM) with bovine pulmonary artery endothelial cells (BPAEC), resulted in its incorporation in PLs with concentration-dependent kinetics. After a 4 h incubation, the proportion of radioactive phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) + phosphatidylinositol (PI) isolated by TLC, was 77.9%, 16.4% and 5.7%, respectively. In PC, [(14)C]12(S)-HETE was incorporated at the position 2 of the glycerol. Three major peaks of radioactive PC were isolated on RP-HPLC which were hydrolysed by phospholipase C (PLC). The resulting diacylglycerols were derivatized and identified by GC/MS as 1-oleyl-, 1-stearoyl- and 1-palmitoyl-2-[12-HETE] PC. BPAEC were incubated with [(14)C]12(S)-HETE (1 microM) before stimulation with bradykinin (1 microM). (A) 1-acyl-2-[12-HETE] diacylglycerols were isolated, derivatized and analysed by MS. We identified a major ion with m/z = 926 that corresponds to the molecular ion of authentic 1-stearoyl-2-12(S)-HETE DAG, and 2 other ions with m/z = 924 and 898 that correspond to the molecular ions of 1-oleyl- and 1-palmitoyl-2-12(S)-HETE DAG, respectively. (B) Radioactive PA was isolated and hydrolysed by alkaline phosphatase. The MS of resulting diacylglycerols identified 1-stearoyl-, 1-oleyl-, and 1-palmitoyl-2-12(S)-HETE phosphatidic acids. The quantities of 12-HETE PA and the 3 major 12-HETE diacylglycerols were shown to increase following bradykinin stimulation. Thus, the incorporation of 12(S)-HETE into PLs results in the production of altered phosphatidic acids and diacylglycerols. The time-course of increases in 1-acyl-2-(12-HETE) phosphatidic acids and 1-acyl-2-(12-HETE) diacylglycerols showed maximal concentrations 1 and 2 min after bradykinin stimulation, respectively, followed by the decrease of both compounds. Propranolol, an inhibitor of PA phosphohydrolase, totally abolished the bradykinin-induced increase in 12-HETE DAG while increasing the magnitude and duration of 12-HETE PA release. The inhibiting effect of propranolol on bradykinin-induced increase of 12-HETE DAG demonstrates that 12-HETE PA is the principal precursor for 12-HETE DAG. This affords a novel method for confirming the major role of phospholipase D in PC metabolic pathways triggered during cell signaling.

Effects of feeding ethyl-dihomo-gamma-linolenate on prostaglandin biosynthesis and platelet aggregation in the rabbit.

1. The ethyl ester of dihomo-gamma-linolenic acid (20:3omega6) (1 g/kg/day) was fed to rabbits for 25 days. Plasma lipids and platelet aggregation were analyzed on day 1, 11, 16, 21 and 26. 2. All plasma lipid classes were greatly enriched with 20:3omega6. Arachidonic acid levels were elevated to a smaller extent. The different platelet phospholipid fractions analyzed were also highly enriched with 20:3omega6, whereas the arachidonic acid content in platelet phospholipids was significantly lower than in control animals. 3. The excretion of 7 alpha-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid, the major urinary metabolite of prostaglandin E1 and E2 was increased 4.6 fold by the treatment. 4. Platelet aggregation in response to ADP, collagen and arachidonic acid did not differ at any time betweeen 20:3omega6 treated rabbits and controls. 5. It is concluded that prostaglandin E biosynthesis can be increased by enriching the prostaglandin precursor pool. Platelet aggregation in vitro is not altered by feeding ethyl 20:3omega6.

Synthesis of prostaglandins by the rat renal papilla in vitro. Mechanism of stimulation by angiotensin II.

1. The biosynthesis of prostaglandins in the rat renal papilla was studied in a whole-cell preparation in vitro. Prostaglandins recovered from the incubation medium were identified by gas chromatography-mass spectrometry as prostaglandin E2 and prostaglandin F2alpha. Quantitative estimates of prostaglandin output were obtained by bioassay and confirmed by selected ion monitoring. 2. Prostaglandin biosynthesis was enhanced by exogenous arachidonic acid and also by triglyceride lipase, indicating that arachidonic acid released from papillary triglycerides is readily available for prostaglandin biosynthesis. 3. Angiotensin II (10--100 ng/ml) stimulated the biosynthesis of both prostaglandin E2 and prostaglandin F2alpha, thus increasing prostaglandin levels in both the incubation medium and the tissues. 4. The mechanism whereby angiotensin II stimulates prostaglandin biosynthesis was investigated using the isotope dilution technique. In the presence of [14-C]-arachidonic acid, angiotensin II stimulated the output of more prostaglandin that had a significantly lower specific activity than the controls. Angiotensin II therefore increased the availability of endogenous, non-labelled substrate for prostaglandin biosynthesis. This conclusion was supported by experiments in which enough arachidonic acid was added to make the kinetics of prostaglandin synthesis zero order. Under such conditions angiotensin II failed to cause any further increase in prostaglandin synthesis. 5. It is concluded that angiotensin II controls prostaglandin biosynthesis in the renal papilla by regulating the availability of free precursor. Possible mechanisms for increased levels of free arachidonic acid could be the activation of a tissue acyl hydrolase or decreased utilization of fatty acids.