Activation of soluble guanylate cyclase by arachidonic acid and 15-lipoxygenase products

The activity of soluble guanylate cyclase can be increased by exposure of the enzyme to arachidonic acid or to some oxidized metabolites of the fatty acid. We have tried to determine whether activation of the enzyme by arachidonate requires that the fatty acid be converted to an oxidized metabolite, either by a possible trace contaminant of a lipoxygenase or by guanylate cyclase itself, which contains a heme moiety. Soluble guanylate cyclase purified from bovine lung was activated 4-6-fold by arachidonic acid. This activation was not dependent on the presence of oxygen in the incubation medium. No detectable metabolites of arachidonic acid were formed during incubation with soluble guanylate cyclase. Addition of soybean lipoxygenase to the incubation did not increase activation by arachidonic acid. The inhibitors of lipoxygenase activity, nordihydroguaiaretic acid and eicosatetraynoic acid, had direct effects on soluble guanylate cyclase and interfered with its activation by arachidonate, whereas another lipoxygenase inhibitor, BW 755 C, did not. The data suggest that arachidonic acid increases the activity of guanylate cyclase by direct interaction with the enzyme rather than by being converted to an active metabolite.     

Effect of arachidonic acid on potato tubers during storage]

Potato (Solanum tuberosum L.) tubers were treated with various concentrations (10(-9) to 10(-4) M) of the biogenic elicitor arachidonic acid during the period of storage (from October to July). The data showed that the resistance-inducing concentration of arachidonic acid was 10(-6) M in autumn and 10(-9) M in spring. Possible causes of the change in the immunizing concentration of arachidonic acid during storage of potato tubers are discussed.     

Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme

The mechanism by which arachidonic acid activates soluble guanylate cyclase purified from bovine lung is partially elucidated. Unlike enzyme activation by nitric oxide (NO), which required the presence of enzyme-bound heme, enzyme activation by arachidonic acid was inhibited by heme. Human but not bovine serum albumin in the presence of NaF abolished activation of heme-containing guanylate cyclase by NO and nitroso compounds, whereas enzyme activation by arachidonic acid was markedly enhanced. Addition of heme to enzyme reaction mixtures restored enzyme activation by NO but inhibited enzyme activation by arachidonic acid. Whereas heme-containing guanylate cyclase was activated only 4- to 5-fold by arachidonic or linoleic acid, both heme-deficient and albumin-treated heme-containing enzymes were activated over 20-fold. Spectrophotometric analysis showed that human serum albumin promoted the reversible dissociation of heme from guanylate cyclase. Arachidonic acid appeared to bind to the hydrophobic heme-binding site on guanylate cyclase but the mechanism of enzyme activation was dissimilar to that for NO or protoporphyrin IX. Enzyme activation by arachidonic acid was insensitive to Methylene blue or KCN, was inhibited competitively by metalloporphyrins, and was abolished by lipoxygenase. Whereas NO and protoporphyrin IX lowered the apparent Km and Ki for MgGTP and uncomplexed Mg2+, arachidonic and linoleic acids failed to alter these kinetic parameters. Thus, human serum albumin can promote the reversible dissociation of heme from soluble guanylate cyclase and thereby abolish enzyme activation by NO but markedly enhance activation by polyunsaturated fatty acids. Arachidonic acid activates soluble guanylate cyclase by heme-independent mechanisms that are dissimilar to the mechanism of enzyme activation caused by protoporphyrin IX.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Activation of guanylate cyclase by arachidonic acid in mammary gland homogenates from mice.

Arachidonic acid stimulated guanylate cyclase activity about two fold in homogenates of mammary glands obtained from midpregnant mice; effects of arachidonic acid were observed during incubation periods between 5 and 20 minutes. Stimulatory effects of arachidonic acid on guanylate cyclase activity were observed when 10 to 100 microgram arachidonic acid was added to the reaction mixtures (150 microliter). When 250 microgram or more arachidonic acid was added to the reaction mixtures, the activity of guanylate cyclase was inhibited. Other fatty acids including linoleic acid, linolenic acid and oleic acid also stimulated guanylate cyclase activity but neither arachidic acid nor stearic acid had an effect. The arachidonic acid stimulation of guanylate cyclase activity was abolished by incubation with indomethacin and aspirin, thus suggesting the arachidonic acid effect may be carried out via the prostaglandins. A variety of prostaglandins, however, at several concentrations did not stimulate guanylate cyclase activity when added to the reaction mixtures. The failure of the prostaglandins to have an effect may be due to several reasons which are discussed.     

Activation of guanylate cyclase by arachidonic acid: In Mammary gland homogenates from mice

Arachidonic acid stimulated guanylate cyclase activity about two fold in homogenates of mammary glands obtained from midpregnant mice; effects of arachidonic acid were observed during incubation periods between 5 and 20 minuted. Stimulatory effects of arachidonic acid on guanylate cyclase activity were observed when 10 to 100 μg arachidonic acid was added to the reaction mixtures (150 μl). When 250 μg or more arachidonic acid was added to the reaction mixtures, the activity of guanylate cyclase was inhibited. Other fatty acids including linoleic acid, linolenic acid and oleic acid also stimulated guanylate cyclase activity but neither arachidic acid nor stearic acid had an effect. The arachidonic acid stimulation of guanylate cyclase activity was abolished by incubation with indomethacin and aspirin, thus suggesting the arachidonic acid effect may be carried out via the prostaglandins. A variety of prostaglandins, however, at several concentrations did not stimulate guanylate cyclase activity when added to the reaction mixtures. The failure of the prostaglandins to have an effect may be due to several reasons which are discussed.     

Relationship between lipemia and sensitivity to arachidonic acid in rabbits]

The hypotensive effect of arachidonic acid in the rabbit increases simultaneously with the fall of the plasmatic unesterified fatty acids (NEFA), after treatment with nicotinic acid or heparin. In the case of nicotinic acid sensitization, which occurs in 10 minutes, the fall of NEFA occurs immediately. The sensitization by heparin appears only after 40 minutes following the injection; during this latency, the NEFA are enhanced; after the fall of NEFA, the rabbit becomes more sensitive to arachidonic acid. Mechanisms of heparinic sensitization are discussed.     

Chemical synthesis of cytochrome P-450-dependent metabolites of arachidonic acid]

Approaches to the chemical synthesis of cytochrome P-450-dependent metabolites of arachidonic acid and the biological role of novel metabolites in the arachidonic acid cascade are discussed.     

Metabolism of [C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

Activation of arachidonic acid metabolism--a protective reaction of the body during irradiation and combined radiation-thermal damage

Metabolic activity of arachidonic acid cascade enzymes in rabbit polymorphonuclear leucocytes sharply rises during the first day of irradiation, decreases at the highest mortality period, and is restored at later times. The inhibitors of the activity of lipoxygenase and cyclooxygenase and the release of arachidonic acid have been shown to increase the mortality rate and to decrease the average lifetime of rats exposed to ionizing radiation and a mixture of radiation and heat.     

Preparation of deuterated arachidonic acid

Arachidonic acid is transformed to a wide variety of oxygenated metabolites. The limiting factor in the quantitation of these metabolites by GC/MS using selected ion monitoring (SIM) is the availability of suitable stable-isotope labelled internal standards. We report a convenient procedure for the preparation of ²H₈-arachidonic acid, from which the desired deuterated metabolite standards can be produced by a combination of chemical and biological procedures.     

Protein kinase C subspecies in adult rat hippocampal synaptosomes. Activation by diacylglycerol and arachidonic acid

Synaptosomes isolated from the adult rat hippocampus contain the alpha- and beta-subspecies of protein kinase C (PKC), but not the gamma-subspecies which is abundantly expressed in the pyramidal cells in this brain region. Although the gamma-subspecies is known to respond significantly to free arachidonic acid, it is found that both the alpha- and beta-subspecies are also activated dramatically by arachidonic acid in synergistic action with diacylglycerol. Oleic, linoleic, and linolenic acids are all active. It is possible that unsaturated fatty acids may take part in the activation of alpha- and beta-subspecies of PKC which are present in the presynaptic nerve endings terminating at the hippocampal pyramidal cells.     

The essentiality of arachidonic acid and docosahexaenoic acid

MCF-10A breast epithelial cells treated with docosahexaenoic acid (DHA) or oleic acid (OA) accumulated cytoplasmic lipid droplets containing both triacylglycerol and cholesteryl esters (CE). Interestingly, total CE mass was reduced in cells treated with DHA compared to cells treated with OA, and the CEs were rich in n-3 fatty acids. Thus, we hypothesized that DHA may be, in addition to a substrate, an inhibitor of cholesterol esterification in MCF-10A cells. We determined that the primary isoform of acyl-CoA: cholesterol acyltransferase expressed in MCF-10A cells is ACAT1. We investigated CE formation with DHA, OA, and the combination in intact cells and isolated microsomes. In both cells and microsomes, the rate of CE formation was faster and more CE was formed with OA compared to DHA. DHA substantially reduced CE formation when given in combination with OA. These data suggest for the first time that DHA can act as a substrate for ACAT1. In the manner of a poor substrate, DHA also inhibited the activity of ACAT1, a universally expressed enzyme involved in intracellular cholesterol homeostasis, in a cell type that does not secrete lipids or express ACAT2.     

Heart arachidonic acid is uniquely sensitive to dietary arachidonic acid and docosahexaenoic acid content in domestic piglets

This study determined the sensitivity of heart and brain arachidonic acid (ARA) and docosahexaenoic acid (DHA) to the dietary ARA level in a dose–response design with constant, high DHA in neonatal piglets. On day 3 of age, pigs were assigned to 1 of 6 dietary formulas varying in ARA/DHA as follows (% fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. At necropsy (day 28) higher levels of dietary ARA were associated with increased heart and liver ARA, while brain ARA remained unaffected. Dietary ARA had no effect on tissue DHA accretion. Heart was particularly sensitive, with pigs in the intermediate groups having different ARA (A2, 18.6±0.7%; A3, 19.4±1.0%) and a 0.17% increase in dietary ARA resulted in a 0.84% increase in heart ARA. Further investigations are warranted to determine the clinical significance of heart ARA status in developing neonates.     

Production of arachidonic acid byMortierella fungi

The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.