Douglas A. Peterson

The non specificity of specific nitric oxide synthase inhibitors

L-NAME (Nw-Nitro-L-arginine methylester) and L-NMMA (NG- Monomethyl-L-arginine, monoacetate) are used widely as nitric oxide (NO) synthase inhibitors. Because of their functional groups (alcohols, amines and carboxylates), it appeared that they could interact with iron in a variety of systems. Using three in vitro models we observed these two compounds had inhibitory effects on cytochrome C reduction by ferrous iron, by ferrous iron accelerated by an unsaturated fatty acid or by epinephrine. This suggests that L-NAME and L-NMMA could have effects in iron containing systems found intracellularly apart from their inhibition of (NO) synthesis.

Phosphatidic acid releases calcium from a platelet membrane fraction in vitro

A platelet membrane fraction which actively sequesters calcium in the presence of ATP was prepared and the influence of phosphatidic acid evaluated. At 10--60 micrograms/ml phosphatidic acid caused a concentration dependent release of calcium from the membrane fraction. The calcium was released from inside the vesicles, since release occurred in the presence of EGTA used to bind calcium outside the membrane vesicles. Aspirin failed to inhibit release of calcium by phosphatidic acid. Our results may explain, in part, the prostaglandin and thromboxane independent calcium release which occurs in response to certain aggregating agents. Thus, phosphatidic acid, or a metabolite, may have an important role intracellularly in platelets in promoting calcium movement.

Pergolide Is an Inhibitor of Voltage-Gated Potassium Channels, Including Kv1.5, and Causes Pulmonary Vasoconstriction

Background—Pergolide produces clinical benefit in Parkinson disease by stimulating dopamine D1 and D2 receptors. An increased incidence of carcinoid-like heart valve disease (CLHVD) has been noted in pergolide users, reminiscent of that induced by certain anorexigens used for weight reduction. Anorexigens that modulate serotonin release and reuptake, such as dexfenfluramine, were withdrawn from sale because of CLHVD. Interestingly, the anorexigens also caused pulmonary arterial hypertension (PAH). Anorexigens were shown to enhance hypoxic pulmonary vasoconstriction, in part by inhibiting voltage-gated K+ channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). Although PAH has not been associated with pergolide use, we hypothesized that pergolide might have similar effects on hypoxic pulmonary vasoconstriction and Kv channels. Methods and Results—Pergolide enhanced hypoxic pulmonary vasoconstriction in the isolated perfused rat lung compared with control lungs (mean pulmonary artery pressure 32±3 versus 21±2 mm Hg; P<0.01). Pergolide also caused vasoconstriction in rat pulmonary artery rings. Pergolide inhibited PASMC potassium current density, resulting in membrane depolarization (from −51±2 to −44±1 mV) and increased cytosolic calcium in both rat and human PASMCs. Pergolide directly inhibited heterologously expressed Kv1.5 and KCa channels. Conclusions—Pergolide causes Kv channel inhibition and, despite being from a different class of drugs, has pulmonary vascular effects reminiscent of dexfenfluramine. Coupled with their shared proclivity to induce CLHVD, these findings suggest that clinical monitoring for pergolide-induced PAH should be considered.

Ferrous iron mediated oxidation of arachidonic acid: studies employing nitroblue tetrazolium (NBT).

The oxidation of arachidonic acid by ferrous sulfate provides a useful model to study the role of iron in lipid oxidation reactions. We have employed nitroblue tetrazolium (NBT) in the present investigation to evaluate the mechanism of this reaction. In the presence of arachidonic acid, Fe +++, and O2, the yellow dye NBT was rapidly reduced to the blue form, NBTH2. The molar ratio of arachidonic acid to Fe++ in this rapid reaction was 1:1, showing an interaction of one fatty acid molecule per iron molecule. Approximately one molecule of NBT was reduced per four molecules of arachidonic acid and Fe++. Reduction of NBT was accompanied by oxidation of Fe++ to Fe+++, suggesting the transfer of four electrons from the Fe++ to NBT to reduce the dye. Arachidonic acid was found to be unchanged when extracted at the end of the reaction, indicating formation of a complex that could dissociate leaving intact arachidonic acid. Evidence for the presence of such a complex which slowly dissociates during the reaction was obtained after longer incubations with small amounts of arachidonic acid. NBT reduction was not inhibited by agents which hydrolyze superoxide, by catalase or by agents which trap hydroxy radicals. We, therefore, propose a model in which NBT traps a radical generated on the arachidonic acid molecule. The proposed model suggests mechanisms for other fatty acid oxidation reactions such as prostaglandin and hydroperoxy fatty acid synthesis.

Polyunsaturated fatty acids stimulate superoxide formation in tumor cells: A mechanism for specific cytotoxicity and a model for tumor necrosis factor?

Some neoplastic cell lines are readily killed when incubated in the presence of polyunsaturated fatty acids (PUFA). In an attempt to elucidate this phenomenon, we studied PUFA-driven superoxide (O2-) production by cultured NS-1 cells (murine lymphoid tumor cells). We find: (1) Even in the absence of added PUFA, NS-1 cells generate O2- (i.e., reduce nitroblue tetrazolium). (2) addition of PUFA increases O2- by greater than 50%. (3) Artificial loading of NS-1 cells with liposome encapsulated superoxide dismutase prevents the majority of spontaneous and PUFA-driven NBT reduction. We conclude that PUFA drives O2- generation by tumor cells, that this generation is largely intracellular, and that this phenomenon may help explain toxicity of PUFA for tumor cells.

Salicylic acid inhibition of the irreversible effect of acetylsalicylic acid on prostaglandin synthetase may be due to competition for the enzyme cationic binding site

Salicylic acid (SA), a weak inhibitor of the prostaglandin endoperoxide synthetase or fatty acid cyclooxygenase enzyme, is known to prevent irreversible enzyme inhibition by acetylsalicylic acid (ASA). The interaction of arachidonic acid with ferrous sulfate was used as a model to study the reaction of the fatty acid with the postulated enzymic cationic binding site on Fe2+-heme. SA was as potent as ASA in inhibiting the cooxygenation of arachidonic acid and ferrous sulfate. The results suggests that SA could complete effectively for the enzyme cationic site with ASA. Thus SA may block ASA acetylation of the cyclooxygenase by preventing ASA from binding to this site.

Inhibition of ferrous iron induced oxidation of arachidonic acid by indomethacin

The molecular mechanism by which indomethacin exerts its inhibitory effects on the prostaglandin endoperoxide synthetase enzyme is unknown. In the present study we have explored the possibility that indomethacin might interact with Fe++ in the enzyme to produce its inhibitory effect. For this study we made use of the recent discovery that Fe++ alone can oxidize arachidonic acid, and the interaction of this fatty acid with the metal can be detected by following reduction of nitroblue tetrazolium (NBT) or by conversion of the Fe++ to Fe+++. Indomethacin markedly inhibited NBT reduction in the presence of arachidonic acid and Fe++ when the indomethacin had been preincubated with the Fe++. Indomethacin also inhibited the conversion of Fe++ to Fe+++ by arachidonic acid. Results obtained by varying the concentrations of indomethacin and arachidonic acid and measuring inhibition of the conversion of Fe++ to Fe+++ by the indomethacin are consistent with a one to one complex forming between indomethacin and Fe++. The complex between indomethacin and Fe++ separates on prolonged incubation of the complex with arachidonic acid. The nature of the binding is suggested by a molecular model. Our results suggest that indomethacin may act to inhibit the prostaglandin endoperoxide synthetase enzyme by complexing Fe++ in the enzyme. Ibuprofen and tolmetin, two other prostaglandin synthetase inhibitors, also inhibit the interaction of Fe++ with arachidonic acid suggesting this may be a general mechanism for this type of drug.

Mechanisms of oxygen sensing : a key to therapy of pulmonary hypertension and patent ductus arteriosus

Specialized tissues that sense acute changes in the local oxygen tension include type 1 cells of the carotid body, neuroepithelial bodies in the lungs, and smooth muscle cells of the resistance pulmonary arteries and the ductus arteriosus (DA). Hypoxia inhibits outward potassium current in carotid body type 1 cells, leading to depolarization and calcium entry through L‐type calcium channels. Increased intracellular calcium concentration ([Ca++]i) leads to exocytosis of neurotransmitters, thus stimulating the carotid sinus nerve and respiration. The same K+ channel inhibition occurs with hypoxia in pulmonary artery smooth muscle cells (PASMCs), causing contraction and providing part of the mechanism of hypoxic pulmonary vasoconstriction (HPV). In the SMCs of the DA, the mechanism works in reverse. It is the shift from hypoxia to normoxia that inhibits K+ channels and causes normoxic ductal contraction. In both PA and DA, the contraction is augmented by release of Ca++ from the sarcoplasmic reticulum, entry of Ca++ through store‐operated channels (SOC) and by Ca++ sensitization. The same three ‘executive’ mechanisms are partly responsible for idiopathic pulmonary arterial hypertension (IPAH). While vasoconstrictor mediators constrict both PA and DA and vasodilators dilate both vessels, only redox changes mimic oxygen by having directly opposite effects on the K+ channels, membrane potential, [Ca++]i and tone in the PA and DA. There are several different hypotheses as to how redox might alter tone, which remain to be resolved. However, understanding the mechanism will facilitate drug development for pulmonary hypertension and patent DA.

The role of ion channels in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which localized flow of blood in small resistance pulmonary arteries is matched to alveolar ventilation. This chapter discusses the role of several potassium and calcium channels in HPV, both in enhancing calcium influx into smooth muscle cells (SMCs) and in stimulating the release of calcium from the sarcoplasmic reticulum, thus increasing cytosolic calcium. The increase in calcium sensitivity caused by hypoxia is reviewed in Chapter 19. Particular attention is paid to the activity of the L-type calcium channels which increase calcium influx as a result of membrane depolarization and also increase calcium influx at any given membrane potential in response to hypoxia. In addition, activation of the L-type calcium channel may, in the absence of any calcium influx, cause calcium release from the sarcoplasmic reticulum. Many of these mechanisms have been reported to be involved in both HPV and in normoxic contraction of the ductus arteriosus.

Epinephrine and other activators of prostaglandin endoperoxide synthetase can reduce Fe3+-heme to Fe2+-heme.

In view of recent evidence that activation of prostaglandin endoperoxide synthetase by lipid peroxides may relate to the ability of such peroxides to reduce heme, we tested other activators of this enzyme. Epinephrine, norepinephrine, ascorbic acid and tryptophan were all found to reduce Fe3+-heme to Fe2+-heme, though tryptophan was considerably weaker than the others. We suggest that reduction of heme by these compounds might account for their ability to reduce the lag phase on addition of substrate to the enzyme. Epinephrine was assessed for its effects on the lag phase in activation of soybean lipoxygenase and was found to cause a similar reduction of the lag phase of this related enzyme. These findings support the concept that reduction of Fe3+-heme to Fe2+-heme is critical to activation of both the prostaglandin endoperoxide synthetase and soybean lipoxygenase enzymes, and that mechanisms involved in regulation of the valence of iron are important for regulating enzyme activity.