Ceil A. Herman

Bisallylic hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450.

Polyunsaturated fatty acids can be oxygenated by cytochrome P450 to hydroxy and epoxy fatty acids. Two major classes of hydroxy fatty acids are formed by hydroxylation of the ω-side chain and by hydroxylation of bisallylic methylene carbons. Bisallylic cytochrome P450-hydroxylases transform linoleic acid to 11-hydroxylinoleic acid, arachidonic acid to 13-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid, 10-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and 7-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and eicosapentaenoic acid to 16-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid, 13-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid and 10-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid as major metabolites. The bisallylic hydroxy fatty acids are chemically unstable and decompose rapidly tocis-trans conjugated hydroxy fatty acids during acidic extractive isolation. Bisallylic hydroxylase activity appears to be augmented in microsomes induced by the synthetic glucocorticoid dexamethasone and by some other agents, but the P450 gene families of these hydroxylases have yet to be determined. The fatty acid epoxides, which are formed by cytochrome P450, are chemically stable, but are hydrolyzed to diols by soluble epoxide hydrolases. Epoxidation of polyunsaturated fatty acids is a prominent pathway of metabolism int he liver and the renal cortex and epoxygenase activity appears to be under homeostatic control in the kidney. Many arachidonate epoxygenases have been identified belonging to the CYP2C gene subfamily. Epoxygenases have also been found in the central nervous system, endocrine organs, the heart and endothelial cells. Epoxides of arachidonic acid have been found to exert pharmacological effects on many cells.

Prostaglandins or prostaglandin like substances are implicated in normal growth and development in oomycetes

The prostaglandin synthesis inhibitors aspirin and indomethacin inhibit the growth of Achlya caroliniana, A. ambisexualis and Saprolegnia parasitica in a dose-related manner. In addition, the inhibitors cause the formation of a characteristic asterisk-shaped colony. This abnormal colony morphology does not appear to be dependent on medium composition, since three different nitrogen and five differentcarbon sources all support the abnormal growth in the presence of 0.1 mM indomethacin. The abnormal colony morphology is the result of abnormal branching. Inhibitor grown colonies are more densely branched than controls, with shorter distances between branches. Inhibited colonies allowed to grow for greater than ten days escape the inhibition and assume a normal gross colony morphology and size, however, they do not reproduce sexually. The addition of 2 micrograms/ml PGF1 alpha to the growth medium partially overcomes the growth inhibition caused by indomethacin. The data suggest a role for prostaglandin or prostaglandin-like compounds in oomycete development.

Catecholamine effects on blood pressure and heart rate in the American bullfrog, Rana catesbeiana

The effects of catecholamines and adrenergic blocking agents were studied in vivo on the blood pressure and heart rate of the unanaesthetized American bullfrog, Rana catesbeiana. Bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. The mean systemic arterial blood pressure prior to the infusion of catecholamines was 18.5 +/- 1.5 mm Hg. Mean preinfusion heart rate was 30.9 +/- 2.0 beats/min. Epinephrine elicited the largest increase in blood pressure, with an accompanying decrease in heart rate. Norepinephrine and phenylephrine were less effective. Isoproterenol was the only catecholamine tested which elevated heart rate in a dose-dependent manner. It had no effect on blood pressure. The beta adrenergic antagonist, propranolol, blocked the increase in heart rate elicited by isoproterenol but had no effect on the blood pressure increases elicited by the other catecholamines. The alpha adrenergic antagonist, phentolamine, partially blocked the blood pressure increase by epinephrine, norepinephrine, and phenylephrine as well as the elevation of heart rate by isoproterenol. Atropine alone elevated heart rate 19 +/- 3 beats/min, and prevented slowing of the heart due to epinephrine, norepinephrine, and phenylephrine. Stimulatory effects of epinephrine on heart rate were observed only after atropine had been administered. Beta adrenergic receptors, therefore, appear to function in heart rate regulation; however, the predominant effect of catecholamines is reflex slowing of the heart due to stimulation of the vagus nerve. In contrast, the alpha receptor, stimulated by epinephrine, appears to be the main adrenergic receptor controlling blood pressure changes.

Endogenous sulfidopeptide leukotriene synthesis and 12-lipoxygenase activity in bullfrog (Rana catesbeiana) erythrocytes

Endogenous leukotriene (LT) synthesis by mammalian inflammatory cells requires both 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein. Other myeloid cells, like erythrocytes, have an incomplete 5-lipoxygenase pathway and synthesize leukotrienes transcellularly. Several studies indicate that sulfidopeptide leukotrienes have important physiological functions in bullfrogs and receptors have been characterized. Calcium ionophore activated bullfrog blood was analyzed by reverse phase-high-performance liquid chromatography (RP-HPLC). Endogenous metabolites consisted of 5-LO products including leukotriene D4. Other metabolites also suggested 12-lipoxygenase activity. Following purification, metabolites from activated erythrocytes were analyzed by RP-HPLC coupled with radioimmunoassay. Erythrocytes demonstrated endogenous synthesis of LTD4 which was inhibited by non-selective (NDGA) and specific (MK886) 5-lipoxygenase inhibitors. Experiments with partially purified erythrocyte cytosol further confirmed 5-LO activity and revealed 12-lipoxygenase activity. HPLC analysis of [1-14C]arachidonic acid labeled metabolites from activated erythrocytes indicates that most of the available substrate is converted to 12-hydroxy-eicosatetraenoic acid (12-HETE). These novel findings indicate that, in contrast to mammals, bullfrog erythrocytes endogenously synthesize LTD4 and large quantities of 12-HETE giving them the potential to contribute directly to inflammatory responses. The evolutionary loss of the nucleus in mammalian erythrocytes appears to be associated with the inability to synthesize leukotrienes endogenously.

Prostaglandins of the one, two, and three series affect blood pressure in the american bullfrog, Rana catesbeiana.

The effects of prostagandins (PGs), and three potential prostaglandin precursors, were studied on blood pressure and heart rate of the American bullfrog, Rana catesbeiana. Bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. The mean systemic arterial blood pressure (SAP) prior to infusion was 20.4 ± 1.1 mm Hg. Mean preinfusion systolic and diastolic pressures were 23.9 ± 1.4 and 17.1 ± 0.9 mm Hg, respectively. Mean preinfusion heart rate was 41.1 ± 0.5 beats/min. Of the PGs tested, PGI2 was a potent hypotensive agent, with effects at 0.03 μg/kg bw. PGE2 was more potent than PGE3, and PGE1. PGA1 and PGA2 were the least potent, and were ineffective at doses below 100 μg/kg bw. PGF2α was the most potent hypertensive agent tested, with thromboxane B2 less potent. All compounds tested elevated heart rate, with PGE2 the most effective. The prostaglandin precursors, eicosatrienoic acid, arachidonic acid, and eicosapentaenoic acid (2000 μg/kg bw) all decreased blood pressure by approximately 25%. The decrease was attenuated by indomethacin (4 mg/kg bw). These results indicate that the bullfrog utilizes all three hypotensive activity. The ability of the bullfrog to utilize several substrates makes it a good choice for comparative studies on prostaglandin synthesis.

Catecholamine effects on blood pressure and heart rate in warm- and cold-acclimated American bullfrogs (Rana catesbeiana)

The effects of epinephrine (E), norepinephrine (NE), phenylephrine (PHE), and isoproterenol (ISO) were studied in vivo on the blood pressure and heart rate of the conscious American bullfrog, Rana catesbeiana. Bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. In warm-acclimated (22 degrees, W-A) bullfrogs, baseline mean systemic arterial blood pressure (SAP) prior to catecholamine infusion was 16.8 +/- 1.1 mm Hg and mean preinfusion heart rate (HR) was 36.4 +/- 2.1 beats per min. In contrast, cold-acclimated bullfrogs (5 degrees, C-A) had baseline SAP values of 7.4 +/- 0.7 mm Hg and HR values of 6.9 +/- 0.3 beats per min. W-A bullfrogs infused with ISO (0.03 to 30 micrograms/kg body wt) demonstrated a dose-related increase in HR, while the HR of C-A bullfrogs did not respond to any tested dose of ISO. ISO administered to W-A bullfrogs exposed to 5 degrees for 60 min produced no significant HR increase. When the animals were returned to 22 degrees, the HR response to ISO was restored. ISO did not affect SAP in either group. E, NE, and PHE increased SAP in both W-A and C-A bullfrogs, with E the most effective. All three catecholamines affected the SAP of C-A animals less than the W-A group. In W-A bullfrogs, E, NE, and PHE decreased HR, while no effect on HR was observed in the C-A group.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombocytes are the predominant source of endogenous sulfidopeptide leukotrienes in the American bullfrog (Rana catesbeiana)

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.

Frogs produce a physiologically active compound from eicosapentaenoic acid

Prostaglandins have been shown to modulate water flow in anuran amphibian urinary bladders. These experiments examined which fatty acid precursor could be metabolized by bladders, and the effect of metabolites on osmotic water flow. Hemibladders were incubated with precursors or prostaglandins (1 microM) and water flow measured. In addition, hemibladders were incubated with 14C-labelled eicosatrienoic, arachidonic, or eicosapentaenoic acid, and products identified by thin layer chromatography. Addition of prostaglandins E1, E2 and I2 inhibited water flow. Eicosatrienoic acid did not affect water flow. Arachidonic acid inhibited basal water flow, an effect which was not completely reversed with the addition of indomethacin. Eicosapentaenoic acid stimulated water flow, and the stimulation was blocked with indomethacin. Frog urinary bladder did not synthesize any prostaglandins from 14C-eicosatrienoic acid. 14C-arachidonic acid was converted into PGE2 and PGD2. 14C-eicosapentaenoic acid was synthesized into compounds, presumably PGE3 and PGD3, with the opposite physiological effects of two-series prostaglandins. The data suggest that effects of prostaglandins on amphibian bladder depend on the substrate which is metabolized.

Prostaglandin synthesis in goldfish heart, Carassius auratus

Potential precursors for prostaglandin (PG) synthesis were measured in goldfish heart and skeletal muscle by gas chromatography. Heart tissue contained docosahexaenoic, arachidonic, eicosapentaenoic, and eicosatrienoic acids in concentrations of 3223 +/- 128, 1216 +/- 7.8, 260 +/- 72.8, and 250 +/- 14 ng/mg wet wt, respectively. 14C-Labeled substrates were examined for their ability to be converted to prostaglandins. Eicosatrienoic and docosahexaenoic acid were not synthesized into prostaglandins, with 66 and 72% of the substrate remaining as free fatty acids, respectively. In contrast, both arachidonic and eicosapentaenoic acids were converted predominantly to PGFs and PGIs. The conversion was time dependent and complete by 30 min. The conversion patterns with eicosapentaenoic acid and arachidonic acid were essentially the same. The data suggest that goldfish cyclooxygenase can utilize two of the four potential substrates for prostaglandin synthesis. As fatty acid levels in fish vary with environmental temperature, substrate availability rather than cyclooxygenase preference may dictate the types of prostaglandins which are produced.

Hormone-dependent lipoxygenase activity in Achlya ambisexualis

Abstract Homogenates of male Achyla ambisexualis oxidize exogenously added [ 14 C]arachidonic acid to an unidentified lipoxygenase product. Synthesis occurs at a rate of 10.6 ± 1.3 μg mg −1 protein 30 min −1 . Activity in homogenates of female mycelium is only 2.1 ± 1.2. Conversion is eliminated by the lipoxygenase inhibitor nordihydroguaiaretic acid (10 −4 M ). Homogenates prepared from the male grown in chemical but not physical contact with female mycelium had decreased lipoxygenase activity (3.1 ± 1.5), suggesting that antheridiol produced by the female decreases lipoxygenase activity in the male. To confirm this, actively growing male cultures were exposed to 10 −9 M 7-deoxy-7-dihydroantheridiol, a stable analog of antheridiol, for 24 h. Homogenates from these cultures also had diminished lipoxygenase activity (2.7 ± 1.0). 7-Deoxy-7-dihydroantheridiol added to the incubation mixture at 10 −9 M had no effect on lipoxygenase activity (9.0 ± 1.8), excluding a direct action of 7-deoxy-7-dihydroantheridiol on the enzyme. These data support the hypothesis that lipoxygenase products are associated with vegetative growth and suggest the antheridiol initiation of reproductive growth suppresses lipoxygenase activity.